Modulators of the P2Y10 receptor useful in altering T lymphocyte function

ABSTRACT

The present invention relates to modulators of P2Y10. Various modulators are disclosed, including an antibody that binds specifically to P2Y10. The present invention also relates to methods of activating resting T lymphocytes and inhibiting the proliferation of activated T lymphocytes. Methods of upregulating trascription of P2Y10 mRNA in a resting T lymphocyte, methods of inducing expression of P2Y10 on the surface of a resting T lymphocyte and methods of activating a T lymphocyte are disclosed. Also disclosed are pharmaceuticals and methods of treating an immune disease.

The present patent application claims the benefit of U.S. ProvisionalPatent Application Serial No. 60/471,135, filed May 15, 2003, thedisclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to method of modulating thebiological activity of a T lymphocyte via a binding event comprising aP2Y10 receptor. More particularly, the present invention relates to amethod of modulating the biological activity of a T lymphocyte via abinding event comprising binding of a P2Y10 receptor by an antibody. Thepresent invention also relates to modulators, including antibodies,adapted to bind a P2Y10 receptor and to modulate T lymphocyte biologicalactivity. Amino Acid Abbreviations Single-Letter Code Three-Letter CodeName A Ala Alanine V Val Valine L Leu Leucine I Ile Isoleucine P ProProline F Phe Phenylalanine W Trp Tryptophan M Met Methionine G GlyGlycine S Ser Serine T Thr Threonine C Cys Cysteine Y Tyr Tyrosine N AsnAsparagine Q Gln Glutamine D Asp Aspartic Acid E Glu Glutamic Acid K LysLysine R Arg Arginine H His Histidine

Functionally Equivalent Codons

Amino Acid Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGUAspartic Acid Asp D GAC GAU Glumatic Acid Glu E GAA GAG PhenylalaninePhe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAUIsoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU Leucine Leu L UUA UUG CUACUC CUG CUU Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S ACG AGUUCA UCC UCG UCU

BACKGROUND OF THE INVENTION

T lymphocytes mediate immune system function by recognizing membraneantigen on antigen-presenting cells, virus-infected cells, cancer cells,and grafts. Binding of antigen to T lymphocytes either causes the cellsto proliferate and secrete various cytokines, which activates an immuneresponse, or causes the T lymphocyte to acquire cytotoxic activity toaid in the immune response.

While essential to the immune response, aberrant T cell responses havebeen implicated in a number of disorders. For instance, autoimmunediseases such as rheumatoid arthritis and multiple sclerosis are thoughtto result, in part, from T cells recognizing self antigens as foreign.Conversely, dampened immune/T cell responses may play a role in somemalignant cancers.

G-protein-coupled receptors (GPCRs) are of great biologicalsignificance, as the malfunction of GPCRs has been implicated in theonset of many diseases, including, but not limited to, Alzheimer's,Parkinson, diabetes, dwarfism, color blindness, retinal pigmentosa andasthma. Also, GPCRs have also been implicated in depression,schizophrenia, sleeplessness, hypertension, anxiety, stress, renalfailure and in several cardiovascular, metabolic, neurologic,oncology-related and immune disorders (Horn & Vriend, (1998) J. Mol.Med. 76: 464-468). GPCRs have also been shown to play a role in HIVinfection (Feng et al. (1996) Science 272:872-877).

GPCRs are integral membrane proteins characterized by the presence ofseven hydrophobic transmembrane domains that together form a bundle ofantiparallel alpha (α) helices. The 7 transmembrane regions aredesignated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. These proteinsrange in size from under 400 to over 1000 amino acids (Strosberg, (1991)Eur. J. Biochem. 196:110; Coughlin (1994) Curr. Opin. Cell Biol.6:191-197). The amino-terminus of a GPCR is extracellular, is ofvariable length, and is often glycosylated. The carboxy-terminus iscytoplasmic and generally phosphorylated. Extracellular loops of GPCRsalternate with intracellular loops and link the transmembrane domains.Cysteine disulfide bridges linking the second and third extracellularloops may interact with agonists and antagonists. The most conserveddomains of GPCRs are the transmembrane domains and the first twocytoplasmic loops. The transmembrane domains account for structural andfunctional features of the receptor. In some G-protein coupledreceptors, the bundle of α helices forms a ligand-binding pocket formedby several G-protein coupled receptor transmembrane domains.

P2Y10 (GenBank Accession Number AF000545; SEQ ID NOS:1 and 2) is aputative 7-transmembrane GPCR that appears to show lymphoid-restrictedexpression (Rao et al. (1999) J Biol. Chem. 274:34245-34252). Indeed,northern blot analysis of RNA showed P2Y10 expression in B and T cellswith undetectable levels in macrophages and fibroblasts. In mice, theP2Y10 promoter contains a PU.1/Spi-B binding site required for P2Y10transcription. Although it is not the present inventors' intention to bebound by any particular theory of operation, because the PU.1 and Spi-Btranscription factors have been implicated in B cell receptor signalingin mice (Rao et al., (1999) J Biol. Chem. 274:34245-34252), the presentinventors speculated there is a connection between B cell receptorsignaling and P2Y10 in humans. The present inventors further speculatedthere is a connection between T cell receptor signaling and P2Y10 inhumans.

Presently, the structure and function of P2Y10 is under investigation.For example, U.S. Pat. No. 5,834,587 to Chan et al. discloses theidentification of the human G-protein-coupled receptor called HLTEX11and the cloning of the gene encoding the same. Human HLTEX11 is alsoknown as P2Y10 and for clarity and consistency, throughout the presentdisclosure the term P2Y10 is used to describe this polypeptide. Asnoted, Chan et al. disclose a nucleic acid sequence encoding a humanP2Y10 polypeptide, as well as an amino acid sequence of the same.However, Chan et al. do not disclose modulating T lymphocyte activity byemploying an antibody directed against a P2Y10 polypeptide having one ormore of the following properties: (a) the ability to activate a restingT lymphocyte; (b) the ability to induce surface expression of P2Y10 on aresting T lymphocyte; (c) the ability to stimulate P2Y10 mRNA expressionin a resting T lymphocyte; and (d) the ability to inhibit proliferationof an activated T lymphocyte.

Thus, although a nucleic acid sequence encoding a human P2Y10polypeptide, as well as an amino acid sequence of a human P2Y10polypeptide, is known, until the present disclosure, a modulator of Tlymphocyte function that interacts with P2Y10 and has differentfunctions depending on the state of the T lymphocyte has not beendescribed. Therefore, what is needed is a modulator of T lymphocytefunction that interacts with a P2Y10 polypeptide, for example ananti-P2Y10 antibody. Such a T lymphocyte modulator could be employed asa therapeutic agent, as a component of a therapeutic agent, as acomponent of a diagnostic method or as a component of a diagnostic ortherapeutic kit. As described more fully herein below, the presentinvention solves this and other problems.

SUMMARY OF THE INVENTION

A P2Y10 modulator is disclosed. In one embodiment, the modulatorassociates specifically with a P2Y10 polypeptide and has one or moreproperties selected from the group consisting of: (a) the ability toactivate a resting T lymphocyte upon association with a P2Y10polypeptide; (b) the ability to induce expression of a P2Y10 polypeptideon the surface of a resting T lymphocyte upon association with a P2Y10polypeptide; (c) the ability to stimulate expression of mRNA encoding aP2Y10 polypeptide in a resting T lymphocyte upon association with aP2Y10 polypeptide; and (d) the ability to inhibit proliferation of anactivated T lymphocyte upon association with a P2Y10 polypeptide. Themodulator can associate specifically with residues 171-191 of a P2Y10polypeptide (RSTDLNNNKSCFADLGYKQMN; SEQ ID NO:3). Additionally, themodulator can comprise an antibody, for example a monoclonal antibody,such as a monoclonal antibody produced by the hybridoma strainidentified as ATCC accession number PTA-3975.

Further, an isolated antibody that associates specifically with a P2Y10polypeptide is disclosed. In one embodiment, the antibody and has one ormore properties selected from the group consisting of: (a) the abilityto activate a resting T lymphocyte upon association with a P2Y10polypeptide; (b) the ability to induce expression of a P2Y10 polypeptideon the surface of a resting T lymphocyte upon association with a P2Y10polypeptide; (c) the ability to stimulate expression of mRNA encoding aP2Y10 polypeptide in a resting T lymphocyte upon association with aP2Y10 polypeptide; and (d) the ability to inhibit proliferation of anactivated T lymphocyte upon association with a P2Y10 polypeptide. Inother embodiments, the antibody can associate specifically with residues171-191 of a P2Y10 polypeptide (SEQ ID NOS:3 and 4) and/or can be amonoclonal antibody, such as a monoclonal antibody produced by thehybridoma strain identified as ATCC accession number PTA-3975.

A method of activating a resting T lymphocyte is also disclosed. In oneembodiment, the method comprises contacting a resting T lymphocyte witha modulator of a P2Y10 polypeptide. In other embodiments, the modulatorassociates specifically with residues 171-191 of a P2Y10 polypeptide(SEQ ID NOS:3 and 4), and/or the modulator is an antibody, for example amonoclonal antibody, and/or is produced by the hybridoma strainidentified as ATCC accession number PTA-3975.

Additionally, a method of inducing expression of a P2Y10 polypeptide onthe surface of a T lymphocyte is disclosed. In one embodiment, themethod comprises contacting a resting T lymphocyte with a modulator ofP2Y10. In other embodiments, the modulator associates specifically withresidues 171-191 of a P2Y 10 polypeptide (SEQ ID NOS:3 and 4), and/orthe modulator is an antibody, for example a monoclonal antibody, and/oris produced by the hybridoma strain identified as ATCC accession numberPTA-3975.

Further, a method of upregulating transcription of P2Y10 mRNA in aresting T lymphocyte is disclosed. In one embodiment, the methodcomprises contacting a T lymphocyte with a modulator of P2Y10. In otherembodiments, the modulator associates specifically with residues 171-191of a P2Y10 polypeptide (SEQ ID NOS:3 and 4), and/or the modulator is anantibody, for example a monoclonal antibody, and/or is produced by thehybridoma strain identified as ATCC accession number PTA-3975.

Continuing, a method of inhibiting proliferation of an activated Tlymphocyte is disclosed. In one embodiment, the method comprisescontacting an activated T lymphocyte with a modulator of a P2Y10polypeptide. In other embodiments, the modulator associates specificallywith residues 171-191 of a P2Y10 polypeptide (SEQ ID NOs:3 and 4),and/or the modulator is an antibody, for example a monoclonal antibody,and/or is produced by the hybridoma strain identified as ATCC accessionnumber PTA-3975.

A method of modulating an immune response is also disclosed. In oneembodiment, the method comprises contacting a resting T lymphocyte witha modulator of P2Y10. In other embodiments, the modulator associatesspecifically with residues 171-191 of a P2Y10 polypeptide (SEQ ID NOS:3and 4), and/or the modulator is an antibody, for example a monoclonalantibody, and/or is produced by the hybridoma strain identified as ATCCaccession number PTA-3975.

Another method of treating an immune disease is disclosed. In oneembodiment, the method comprises administering a modulator of a P2Y10polypeptide, wherein the modulator has one or more properties selectedfrom the group consisting of: (a) the ability to activate a resting Tlymphocyte; (b) the ability to induce expression of a P2Y10 polypeptideon the surface of a resting T lymphocyte; (c) the ability to stimulateexpression of mRNA encoding a P2Y10 polypeptide in a resting Tlymphocyte; and (d) the ability to inhibit proliferation of an activatedT lymphocyte. In other embodiments, the modulator associatesspecifically with residues 171-191 of a P2Y10 polypeptide (SEQ ID NOS:3and 4), and/or the modulator is an antibody, for example a monoclonalantibody, and/or is produced by the hybridoma strain identified as ATCCaccession number PTA-3975. The immune disease can be selected from therepresentative, but non-limiting, group consisting of rheumatoidarthritis, psoriatic arthritis, multiple sclerosis, juvenile diabetes,asthma, inflammatory bowel disease (such as Crohn's disease andulcerative colitus), pyoderma gangrenum, lupus (systemic lupuserythematosis), myasthenia gravis, psoriasis, dermatitis,dermatomyositis; eczema, seborrhoea, pulmonary inflammation, eyeuveitis, hepatitis, Grave's disease, Hashimoto's thyroiditis, autoimmunethyroiditis, Behcet's or Sjorgen's syndrome (dry eyes/mouth), perniciousor immunohaemolytic anaemia, atherosclerosis, Addison's disease(autoimmune disease of the adrenal glands), idiopathic adrenalinsufficiency, autoimmune polyglandular disease (also known asautoimmune polyglandular syndrome), glomerulonephritis, scleroderma,morphea, lichen planus, viteligo (depigmentation of the skin), alopeciaareata, autoimmune alopecia, autoimmune hypopituatarism, Guillain-Barresyndrome, and alveolitis; T-cell mediated hypersensitivity diseases,including contact hypersensitivity, delayed-type hypersensitivity,contact dermatitis (including that due to poison ivy), uticaria, skinallergies, respiratory allergies (hayfever, allergic rhinitis) andgluten-sensitive enteropathy (Celiac disease); inflammatory diseasessuch as osteoarthritis, acute pancreatitis, chronic pancreatitis,asthma, acute respiratory distress syndrome, Sezary's syndrome andvascular diseases which have an inflammatory and or a proliferatorycomponent such as restenosis, stenosis and artherosclerosis.Inflammatory or immune associated diseases or disorders also includes,but is not limited to: endocrine disorders, rheumatic disorders,collagen diseases, dermatologic disease, allergic disease, opthalmicdisease, respiratory disease, hematologic disease, gastrointestinaldisease, inflammatory disease, autoimmune disease, congenital adrenalhyperplasia, nonsuppurative thyroiditis, hypercalcemia associated withcancer, psoriatic arthritis, rheumatoid arthritis, including juvenilerheumatoid arthritis, ankylosing spondylitis, acute and subacutebursitis, acute nonspecific tenosynovitis, acute gouty arthritis,post-traumatic osteoarthritis, synovitis of osteoarthritis,epicondylitis, systemic lupus erythematosus, acute rheumatic carditis,pemphigus, bullous dermatitis herpetiformis, severe erythema multiforme,exfoliative dermatitis, psoriasis, seborrheic dermatitis, seasonal orperennial allergic rhinitis, serum sickness, bronchial asthma, contactdermatitis, atopic dermatitis, drug hypersensitivity reactions, allergicconjunctivitis, keratitis, herpes zoster ophthalmicus, iritis andiridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis,fulminating or disseminated pulmonary tuberculosis chemotherapy,idiopathic thrombocytopenic purpura in adults, secondarythrombocytopenia in adults, acquired (autoimmune) hemolytic anemia,leukemias and lymphomas in adults, acute leukemia of childhood,ulcerative colitis, regional enteritis, autoimmune vasculitis, multiplesclerosis, myasthenia gravis, anklyosing spondylitis, chronicobstructive pulmonary disease, solid organ transplant rejection, sepsis,and allergy.

Additionally, a pharmaceutical composition is disclosed. In oneembodiment, the pharmaceutical composition comprises a modulator of aP2Y10 polypeptide that has one or more of the following properties: (a)the ability to activate a resting T lymphocyte; (b) the ability toinduce surface expression of P2Y10 on a resting T lymphocyte; (c) theability to stimulate P2Y10 mRNA expression in a resting T lymphocyte;and (d) the ability to inhibit proliferation of an activated Tlymphocyte. In other embodiments, the modulator associates specificallywith residues 171-191 of a P2Y10 polypeptide (SEQ ID NOS:3 and 4),and/or the modulator is an antibody, for example a monoclonal antibody,and/or is produced by the hybridoma strain identified as ATCC accessionnumber PTA-3975.

A method of identifying a modulator of a P2Y10 polypeptide is alsodisclosed. In one embodiment, the method comprises (a) measuring theability of a test molecule to bind to a P2Y10 polypeptide; and (b)measuring one or more of: (i) the ability of said test molecule toactivate a resting T lymphocyte; (ii) the ability of said test moleculeto induce surface expression of P2Y10 on a T lymphocyte; (iii) theability of said test molecule to stimulate P2Y10 mRNA expression in a Tlymphocyte; or (iv) the ability of said test molecule to inhibitproliferation of an activated T lymphocyte; whereby a test molecule isidentified as a modulator if the test molecule binds to a P2Y10polypeptide and exhibits one or more abilities selected from the groupconsisting of (i) to (iv). In other embodiments, the modulatorassociates specifically with residues 171-191 of a P2Y10 polypeptide(SEQ ID NOS:3 and 4), and/or the modulator is an antibody, for example amonoclonal antibody, and/or is produced by the hybridoma strainidentified as ATCC accession number PTA-3975.

The present invention also relates to fusion proteins comprising P2Y10or a modulator of P2Y10 and another protein. For example, a fusionprotein of the present invention can comprise a P2Y10 protein and a Fcregion from an immunoglobulin, such as IgG or a fusion comprising aP2Y10 modulator and an IgG. Such fusion proteins can impart or enhance aproperty of P2Y10 or a P2Y10 modulator, for example solubility,bioavailability or biotolerance.

Additionally, a hybridoma strain identified as ATCC accession numberPTA-3975 is disclosed. The hybridoma can be employed to produce anantibody of the present invention.

Accordingly, it is an object of the present invention to provide amethod of modulating T lymphocyte function. This object is achieved inwhole or in part by the present invention.

An object of the invention having been stated hereinabove, other objectswill be evident as the description proceeds, when taken in connectionwith the accompanying Drawings and Examples as described hereinbelow.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a photograph depicting a western blot of human peripheralblood B cells using anti-P2Y 10 monoclonal antibody.

FIG. 2 is a photograph depicting the effect of T cell stimulation onP2Y10 mRNA levels as determined by RT-PCR. P2Y10 primers were used inlanes 1-6, GAPDH primers in lanes 7-12. The treatments were: Lanes 1 &7, unstimulated cells; lanes 2 & 8, anti-P2Y10-treated cells (1 μg/mL);lanes 3 & 9, anti-P2Y10-treated cells (10 μg/mL); lanes 4 & 10, phorbolester-treated cells; lanes 5 & 11, anti-CD3-stimulated cells; lanes 6 &12, anti-CD3/CD28-stimulated cells.

DETAILED DESCRIPTION OF THE INVENTION

Since P2Y10 showed a lymphoid-restrictive expression pattern, in oneaspect of the present invention, the role of P2Y10 in the proliferationof T cells when activated through the T cell receptor was investigated.In another aspect of the present invention a P2Y10-specific monoclonalantibody that modulates T cell function was generated and identified.The results of these and other experiments that form aspects of thepresent invention indicate that modulators of P2Y10 function might beemployed in the diagnosis and/or treatment of a wide range of disordersof the immune system. These and other aspects of the present inventionare described more fully below.

I. Definitions

All scientific and technical terms used in this application havemeanings commonly used in the art unless otherwise specified. As used inthis application, the following words or phrases have the meaningsspecified.

Following long-standing patent law convention, the terms “a” and “an”mean “one or more” when used in this application, including the claims.

As used herein, the term “about,” when referring to a value or to anamount of mass, weight, time, volume, concentration or percentage ismeant to encompass variations of ±20% or less (e.g., ±15%, ±10%, ±7%,±5%, ±4%, ±3%, ±2%, ±1%, or ±0.1%) from the specified amount, as suchvariations are appropriate.

As used herein, the term “activated,” when used in reference to a Tlymphocyte means a T lymphocyte that is in a state of clonal expansionor clonal anergy or has been stimulated to proceed toward a state ofclonal anergy or clonal expansion or is in a state in which it iscapable of clonal expansion.

As used herein, the terms “agonist” and “activator” are synonymous andrefer to an agent that initiates, supplements or potentiates thebioactivity of a functional P2Y10 gene or protein, or that supplementsor potentiates the bioactivity of a naturally occurring or engineeredfunctional P2Y10 gene or protein. An agonist can be a ligand. Further,an agonist can act by preventing an antagonist from acting on a givenprotein.

As used herein, the terms “amino acid,” “amino acid residue” and“residue” are used interchangeably and mean any of the twenty naturallyoccurring amino acids. An amino acid is formed upon chemical digestion(hydrolysis) of a polypeptide at its peptide linkages. The amino acidresidues described herein are preferably in the “L” isomeric form.However, residues in the “D” isomeric form can be substituted for anyL-amino acid residue, as long as the desired functional property isretained by the polypeptide. NH₂ refers to the free amino group presentat the amino terminus of a polypeptide. COOH refers to the free carboxygroup present at the carboxy terminus of a polypeptide. In keeping withstandard polypeptide nomenclature, abbreviations for amino acid residuesare shown in tabular form presented herein above.

It is noted that the amino acid residue sequences represented herein byformulae have a left-to-right orientation, in the conventional directionof amino terminus to carboxy terminus. In addition, the phrases “aminoacid” and “amino acid residue” are broadly defined to include modifiedand unusual amino acids.

Furthermore, it is noted that a dash at the beginning or end of an aminoacid residue sequence indicates a peptide bond to a further sequence ofone or more amino acid residues, a covalent bond to an amino-terminalgroup, such as NH₂, to an acetyl group or to a carboxy-terminal group,such as COOH.

As used herein, the term “antagonist” and “inhibitor” are synonymous andrefer to an agent that decreases or inhibits the bioactivity of afunctional P2Y10 gene or protein, or that decreases or inhibits thebioactivity of a naturally occurring or engineered P2Y10 gene orprotein. An antagonist can be a ligand. Further, an antagonist can actby preventing an agonist from acting on a given protein.

As used herein, the term “antibody” means polyclonal, monoclonal,antibody fragments and antibody derivatives. The term encompassesantibodies prepared by recombinant techniques, such as chimeric orhumanized antibodies, as well as single chain or bispecific antibodies.The term specifically encompasses antibodies that bind to an epitope, ora portion thereof, that is recognized by an antibody described in thepresent invention and/or is secreted by hybridoma strain PTA-3975.

As used herein, the terms “antigen” and “epitope,” which are wellunderstood in the art, mean all or a portion of a macromolecule that isspecifically recognized by a component of the immune system, e.g., anantibody or a T-cell antigen receptor. An epitope is a region of anantigen. As used herein, the term “antigen” encompasses antigenicepitopes, e.g., fragments of an antigen that are antigenic epitopes.

As used herein, the terms “associate” and “bind,” and grammaticalderivations thereof, are used interchangeably and mean a condition ofproximity between or amongst molecules, structural elements, chemicalcompounds or chemical entities. An association can be non-covalent(i.e., reversible), wherein the juxtaposition is energetically favoredby hydrogen bonding or van der Waals or electrostatic interactions, orit can be covalent (i.e., irreversible). Thus in the present disclosure,when it is stated that a ligand “associates” with or “binds” to aprotein, it is meant that the ligand interacts with the protein viacovalent or non-covalent interactions. In one embodiment the ligand canbe an antigen or epitope and the protein can be an antibody. In arelated aspect, the term “associates specifically,” and grammaticalderivations thereof, means an interaction between a first moiety (e.g. amodulator) and a second moiety (e.g., a P2Y10 polypeptide or fragmentthereof) that occurs preferentially to an interaction the first orsecond moiety and any other moieties present. For example, an antibodyis presented with a variety of antigens, but only binds to a particularantigen. In this example, the antibody “specifically associates” withthe particular antigen.

As used herein, the terms “bioactivity” and “biological activity” areused interchangeably and mean any observable effect flowing frominteraction between a P2Y10 polypeptide and a ligand, including anantibody. Representative, but non-limiting, examples of biologicalactivity in the context of the present invention include (a) the abilityto activate a resting T lymphocyte, (b) the ability to induce expressionof a P2Y10 polypeptide on the surface of a resting T lymphocyte, (c) theability to stimulate expression of mRNA encoding a P2Y10 polypeptide ina resting T lymphocyte, and (d) the ability to inhibit proliferation ofan activated T lymphocyte and modulating an immune response.

As used herein, the terms “chimeric protein” and “fusion protein’ areused interchangeably and mean a fusion of a first molecule (e.g., aP2Y10 polypeptide or a modulator) with an amino acid sequence of secondpolypeptide molecule (e.g., an IgG Fc domain). In general, a chimeric orfusion protein of the present invention can be represented by thegeneral formula X—P2Y10 polypeptide or modulator—Y, wherein modulatorrepresents a modulator of the present invention, and X and Y areindependently absent or represent the same or different molecules. Inone embodiment, a chimeric protein comprises an IgG Fc domain fused to amodulator of the present invention. As discussed herein, a modulator ofthe present invention can be a small molecule, polypeptide or otherstructure. When the modulator is a polypeptide, such as a P2Y10polypeptide, the fusion protein can be expressed from a single chimericgene encoding the chimeric protein.

As used herein the term “complementary” means a nucleic acid sequencethat is base paired, or is capable of base-pairing, according to thestandard Watson-Crick complementarity rules. These rules generally holdthat guanine pairs with cytosine (G:C) and adenine pairs with eitherthymine (A:T) in the case of DNA, or adenine pairs with uracil (A:U) inthe case of RNA.

As used herein, the term “detecting” means confirming the presence of atarget entity by observing the occurrence of a detectable signal, suchas a radiologic, fluorescent, colorimetric, etc. signal that will appearexclusively in the presence of the target entity.

As used herein, the term “diagnosing” means determining the presence orabsence of a condition in a subject. Although a subject is preferably amammal, and more preferably a human, a subject can be any livingentitiy, including individual cells, such as PBMCs. Further, in thecontext of the present invention, the term “condition” is used broadlyand refers to any identifiable state, but preferably refers to a diseasestate, such as an immune disorder.

As used herein, the term “immune response” means a humoral and/or acellular immune response. More particularly, a “humoral immune response”refers to an immune response mediated by antibody molecules, while a“cellular immune response” is one mediated by T-lymphocytes and/or otherwhite blood cells.

Continuing, an immune response can be one that stimulates the productionof cytotoxic T lymphocytes (CTLs), and/or the production or activationof helper T-cells and/or the elicitation of an antibody-mediated immuneresponse. Hence, an immune response can include one or more of thefollowing effects: the production of antibodies by, e.g., but notlimited to B-cells; and/or the activation of suppressor T-cells and/oractivation of γδ T-cells directed specifically to an antigen or antigenspresent in the composition or vaccine of interest.

As used herein, the terms, “increasing,” “inducing,” and “enhancing,”and grammatical derivations thereof, are used interchangeably hereinwith reference to an immune response (e.g., a CTL response or a humoralresponse), and refer to any increase in an immune response overbackground; the terms include inducing an immune response over anabsence of a measurable immune response, or increasing immune responseover a previously measurable immune response.

As used herein, the term “inhibit” and grammatical derivations thereof,means to decrease, limit, or block an action or function. For example,the term can be applied to T lymphocyte activation, in which usage itmeans to decrease, limit or block activation of a T lymphocyte.

As used herein, the terms “isolated” and “purified” are usedinterchangeably and refer to material (e.g., a nucleic acid or apolypeptide) removed from its original environment (e.g., the naturalenvironment, if it is naturally occurring), and thus is altered “by thehand of man” from its natural state. For example, an isolatedpolynucleotide could be part of a vector or a composition of matter, orcould be contained within a cell, and still be “isolated” because thatvector, composition of matter, or particular cell is not the originalenvironment of the polynucleotide. The term “isolated” does not refer togenomic or cDNA libraries, whole cell total or mRNA preparations,genomic DNA preparations (including those separated by electrophoresisand transferred onto blots), sheared whole cell genomic DNA preparationsor other compositions where the art demonstrates no distinguishingfeatures of the polynucleotide and/or protein sequences of the presentinvention; such sequences are excluded from the scope of the presentinvention.

As used herein, the term “ligand” means any molecule that is known orsuspected to associate with another molecule. The term “ligand”encompasses inhibitors, activators, agonists, antagonists, naturalsubstrates and analogs of natural substrates. A ligand can comprise, forexample, a nucleic acid sequence, an amino acid sequence (e.g. a peptideand/or polypeptide) or a small molecule.

As used herein the term “modulate,” and grammatical derivations thereof,refer to an increase, decrease, or other alteration of any and/or allchemical and/or biological activities or properties mediated by a givenDNA sequence, RNA sequence, polypeptide, peptide or molecule. Thedefinition of “modulator” as used herein encompasses agonists and/orantagonists of a particular activity or protein. The term “modulate”therefore refers to both upregulation (i.e., activation or stimulation)and downregulation (i.e. inhibition or suppression) of a response by anymode of action. In one embodiment, a modulator modulates protein (e.g.,P2Y10) activity. In another embodiment, a modulator modulatestranscription of a gene (e.g. a P2Y10 gene).

As used herein, the terms “organism”, “subject” and “patient” are usedinterchangeably and mean any organism referenced herein, includingprokaryotes, though the terms preferably refer to eukaryotic organisms,notably mammals (e.g., mice, rats, dogs and pigs), but most preferablyto humans. The methods of the present invention are particularly usefulin the treatment and diagnosis of warm-blooded vertebrates.

As used herein, the terms “P2Y10 gene” and “recombinant P2Y10 gene” meana nucleic acid molecule comprising an open reading frame encoding aP2Y10 polypeptide of the present invention, including both exon and(optionally) intron sequences.

As used herein, the terms “P2Y10 gene product”, “P2Y10 protein”, “P2Y10polypeptide”, “P2Y10 polypeptide gene product” and “P2Y10 peptide” areused interchangeably and mean peptides having amino acid sequences whichare substantially identical to native amino acid sequences from anorganism of interest and which are biologically active in that theycomprise all or a part of the amino acid sequence of a P2Y10polypeptide, or cross-react with antibodies raised against a P2Y10polypeptide. Such biological activity can include immunogenicity. In oneembodiment, a “P2Y10 gene product”, “P2Y10 protein”, “P2Y10polypeptide”, “P2Y10 polypeptide gene product” or “P2Y10 peptide” isencoded by the sequence of GenBank Accession Number AF000545 (SEQ IDNO:1) and has the amino acid sequence of SEQ ID NO:2.

As used herein, the terms “P2Y10 gene product”, “P2Y10 protein”, “P2Y10polypeptide”, “P2Y10 polypeptide gene product” and “P2Y10 peptide” alsoinclude analogs of a P2Y10 polypeptide. By “analog” is intended that aDNA or peptide sequence can contain alterations relative to thesequences disclosed herein, yet retain all or some of the biologicalactivity of those sequences. Analogs can be derived from genomicnucleotide sequences as are disclosed herein or from other organisms, orcan be created synthetically. Those skilled in the art will appreciatethat other analogs, as yet undisclosed or undiscovered, can be used todesign and/or construct P2Y10 analogs. There is no need for a “P2Y10gene product”, “P2Y10 protein”, “P2Y10 polypeptide”, or “P2Y10 peptide”to comprise all or substantially all of the amino acid sequence of afull length P2Y10 polypeptide gene product.

The terms a “P2Y10 gene product”, “P2Y10 protein”, “P2Y10 polypeptide”,and “P2Y10 peptide” encompass sequences comprising one or moreconservative substitutions in the P2Y10 amino acid sequence of SEQ IDNO:2. The substititution can be naturally occurring or introduced byman. In a conservative substitution, the replacement group will haveapproximately the same size, shape, hydrophobicity and charge as theoriginal group. A table disclosing some representative, but non-limitingproperties that can be used as a guide when identifying or generating aconservative mutation follows:

Representative Conservative Amino Acid Substitutions

-   -   Amino Acid Property Amino Acid        -   Basic: arginine            -   lysine            -   histidine        -   Acidic: glutamic acid            -   aspartic acid        -   Polar: glutamine            -   asparagine        -   Hydrophobic: leucine            -   isoleucine            -   valine        -   Aromatic: phenylalanine            -   tryptophan            -   tyrosine        -   Small: glycine            -   alanine            -   serine            -   threonine            -   methionine

Conservative substitutions typically include the substitution of oneamino acid for another with similar characteristics, e.g., substitutionswithin the following groups: valine, glycine; glycine, alanine; valine,isoleucine, leucine; aspartic acid, glutamic acid; asparagine,glutamine; serine, threonine; lysine, arginine; and phenylalanine,tyrosine. Other conservative amino acid substitutions can be taken fromthe table below. For Amino Acid Code Replace with any of: Alanine AD-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys,homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine ND-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic Acid D D-Asp, D-Asn,Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr,D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp GlutamicAcid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala,Pro, D-Pro, .beta.-Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu,Met, D-Met Leucine L D-Leu, Val, D-Val, Met, D-Met Lysine K D-Lys, Arg,D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-OrnMethionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-ValPhenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp,Trans-3,4, or 5-phenylproline, cis-3,4, or 5-phenylproline Proline PD-Pro, L-1-thioazolidine-4-carboxylic acid, D- orL-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr,Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr, Ser,D-Ser, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Tyrosine YD-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-Leu, Ile,D-Ile, Met, D-Met

The terms a “P2Y10 gene product”, “P2Y10 protein”, “P2Y10 polypeptide”,and “P2Y10 peptide” encompass natural variants of a P2Y10 polypeptide(e.g. SEQ ID NO:2). As used herein, the term “natural variant” means aform of P2Y10 that comprises more or fewer amino acids than the sequenceof SEQ ID NO:2, however in a natural variant the addition or deletion ofamino acids from the sequence of SEQ ID NO:2 is a natural event andoccurs in vivo without intervention by man. For example a P2Y10 naturalvariant is the result of a naturally occurring splicing event thatoccurs in the process of transcribing a P2Y10 polypeptide-encodingsequence (e.g., SEQ ID NO:1). Thus the terms encompass allelic variants,genetically altered versions of the gene, etc.

Shorter or longer sequences are anticipated to be of use in the presentinvention; shorter sequences are herein referred to as “segments”. Insome embodiments, a shorter or longer sequence derived from a P2Y10polypeptide retains the biological activity of a full length P2Y10polypeptide, namely (a) the ability to activate a resting T lymphocyte,(b) the ability to induce expression of a P2Y10 polypeptide on thesurface of a resting T lymphocyte, (c) the ability to stimulateexpression of mRNA encoding a P2Y10 polypeptide in a resting Tlymphocyte, and (d) the ability to inhibit proliferation of an activatedT lymphocyte and modulating an immune response.

Thus, the terms “P2Y10 gene product”, “P2Y10 protein”, “P2Y10polypeptide”, and “P2Y10 peptide” also include fusion, chimeric orrecombinant P2Y10 polypeptides and proteins comprising sequences of thepresent invention. Methods of preparing such proteins are disclosedherein and are known in the art.

As used herein, the terms “protein”, “polypeptide” and “peptide” areused interchangeably and mean any polymer comprising any of the 20protein amino acids, regardless of its size. Although “protein” is oftenused in reference to relatively large polypeptides, and “peptide” isoften used in reference to small polypeptides, usage of these terms inthe art overlaps and varies. Therefore, term “polypeptide” as usedherein refers to peptides, polypeptides and proteins, unless otherwisenoted. Further, the terms “protein”, “polypeptide” and “peptide” areused interchangeably herein.

Thus, a polypeptide of the present invention can comprise amino acidsjoined to each other by peptide bonds or modified peptide bonds, i.e.,peptide isosteres, and can contain amino acids other than the 20gene-encoded amino acids. A polypeptide can be modified by eithernatural processes, such as by posttranslational processing, or bychemical modification techniques which are known in the art. Suchmodifications will be known to those of ordinary skill in the art.Modifications can occur anywhere in a polypeptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.The same type of modification can be present in the same or varyingdegrees at several sites in a given polypeptide.

Also, a given polypeptide can contain many types of modifications. Apolypeptide can be branched, for example, as a result of ubiquitination,or a polypeptide can be cyclic, with or without branching. Cyclic,branched, and branched cyclic polypeptides can result fromposttranslation natural processes or can be made by synthetic methods.Representative modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of phosphotidylinositol, cross-linking,cyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristoylation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, transfer-RNA mediated additionof amino acids to proteins such as arginylation, and ubiquitination(see, e.g., Creighton, Proteins—Structure And Molecular Properties, 2nded., W. H. Freeman and Company, New York, N.Y., USA (1993);Posttranslational Covalent Modification Of Proteins, (Johnson, ed.),Academic Press, New York, N.Y., USA, pp. 1-12 (1983); Seifter et al.,(1990) Method Enzymol. 182:626-646; Rattan et al., (1992) Ann. N.Y.Acad. Sci. 663:48-62, incorporated herein by reference).

As used herein, the term “polypeptide fragment” means an amino acidsequence that is at least one amino acid shorter than a referencesequence, but retains the order of amino acids in the referencesequence. For example, a “polypeptide fragment” of a P2Y10 polypeptidemeans an amino acid sequence that is at least 338 amino acids in length(one residue less than the 339 residues shown in SEQ ID NO:2). Inanother example, a polypeptide fragment of a P2Y10 polypeptide comprisesresidues 171-191 (SEQ ID NOS:3 and 4) of a P2Y10 polypeptide (SEQ IDNO:2).

As used herein, the term “resting” when used in reference to a Tlymphocyte means a T lymphocyte that is not in a state of clonalexpansion or clonal anergy or that has not been stimulated to proceedtoward a state of clonal expansion or clonal anergy or that is in astate in which it is not capable of clonal expansion.

As used herein, the term “small molecule” means any molecule having amolecular weight of 5000 Daltons or less.

As used herein, the term “substantially identical” means at least 70%sequence identity between two amino acid sequences. Sequence identity iscalculated based on a reference sequence, which can be a subset of alarger sequence. A reference sequence will usually be at least about 6amino acids long or more usually at least about 10 amino acids long, andcan extend to the complete sequence that is being compared. Algorithmsfor sequence analysis are known in the art, such as BLAST, described inAltschul et al. (1990) J. Mol. Biol. 215: 403-10.

The term “similarity” is contrasted with the term “identity”. Similarityis defined as above; “identity”, however, means an amino acid sequencehaving the same amino acid at the same relative position in a givenfamily member of a gene family. Homology and similarity are generallyviewed as broader terms than the term identity. Biochemically similaramino acids, for example leucine/isoleucine or glutamate/aspartate, canbe present at the same position—these are not identical per se, but arebiochemically “similar.” As disclosed herein, these are referred to asconservative differences or conservative substitutions. This differsfrom a conservative mutation at the DNA level, which changes thenucleotide sequence without making a change in the encoded amino acid,e.g. TCC to TCA, both of which encode serine.

Polypeptides that are substantially identical to a P2Y10 polypeptide(SEQ ID NO:2) can have between about 70% and 80%, preferably betweenabout 81% to about 90% or even more preferably between about 91% and 99%sequence identity with the corresponding sequence of the native P2Y10protein. Sequences having lesser degrees of identity but comparablebiological activity are considered to be equivalents.

As used herein, the term “T lymphocyte” means any immune system cellthat originates in the thymus, including cytotoxic cells, helper celland suppressor cells in general, and CD4+ and CD8+ cells in particular.Such cells are normally, but not necessarily, capable of producing oneor more cytokines.

As used herein, the term “vector” means a replicon, such as plasmid,phage or cosmid, to which another DNA segment may be attached so as tobring about the replication of the attached segment.

II. Description of Tables

Table 1 is a table showing P2Y10 surface expression on stimulated Tcells as measured by FACS.

Table 2 is a table showing the effect of anti-P2Y10 on T cellproliferation.

Table 3 is a table showing the effect of antigenic peptide on anti-P2Y10inhibition of anti-CD3-induced T cell proliferation.

III. Antibodies of the Present Invention

In one aspect, the present invention relate to antibodies thatassociates specifically with a polypeptide, polypeptide fragment, orvariant of a P2Y10 polypeptide, and/or a P2Y10 epitope (as can bedetermined by immunoassays known in the art for assaying specificantibody-antigen binding). Antibodies of the present invention include,but are not limited to, polyclonal, monoclonal, monovalent, bispecific,heteroconjugate, multispecific, human, humanized or chimeric antibodies,single chain antibodies, Fab fragments, F(ab′) fragments, fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies (including, e.g., anti-Id antibodies to antibodies of thepresent invention), and epitope-binding fragments of any of thepolypeptides and peptides disclosed herein.

The antibodies of the present invention can be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies canbe specific for different epitopes of a P2Y10 polypeptide of the presentinvention or can be specific for both a P2Y10 polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT Publications WO93/17715; WO 92/08802; WO 91/00360; WO 92/05793; U.S. Pat. Nos.4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al.,(1992) J. Immunol. 148:1547-1553; Tutt et al. (1991) J. Immunol.147:60-69.

Antibodies of the present invention can be described or specified interms of an epitope(s) or portion(s) of a polypeptide that a givenantibody recognizes or specifically binds (e.g., residues 171-191 of aP2Y10 polypeptide; SEQ ID NOS:3 and 4). The epitope(s) or polypeptideportion(s) can be specified as described herein, e.g., by N-terminal andC-terminal positions, by size in contiguous amino acid residues, orlisted in the Tables, Figures and/or Sequence Listing. Therefore, thepresent invention includes antibodies that specifically bind P2Y10peptides and polypeptides.

Antibodies of the present invention can also be described in terms oftheir properties, such as their effect on the activation of a resting Tlymphocyte and the inhibition of the proliferation of an activated Tlymphocyte. More particularly, an antibody of the present invention hasone or more of the following properties: (a) the ability to activate aresting T lymphocyte; (b) the ability to induce surface expression ofP2Y10 on a resting T lymphocyte; (c) the ability to stimulate P2Y10 mRNAexpression in a resting T lymphocyte; and (d) the ability to inhibitproliferation of an activated T lymphocyte. These properties aredescribed further throughout the present disclosure.

Antibodies of the present invention can also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homologue of a P2Y10 polypeptide of thepresent invention are included in the scope of the present invention.Antibodies that bind a P2Y10 polypeptide (e.g., sSEQ ID NO:2) with atleast 95%, at least 90%, at least 85%, at least 80%, at least 75%, atleast 70%, at least 65%, at least 60%, at least 55%, and at least 50%identity (as calculated using methods known in the art and/or asdescribed herein) or to a P2Y10 polypeptide or fragment (e.g., SEQ IDNOS:3 and 4) are also encompassed by the present invention. In specificembodiments, antibodies of the present invention cross-react withmurine, rat and/or rabbit homologues of human proteins and thecorresponding epitopes thereof.

In a specific embodiment, the above-described cross-reactivity is withrespect to any single specific antigenic or immunogenic P2Y10polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specificantigenic and/or immunogenic P2Y10 polypeptides disclosed herein.Antibodies of the present invention can also be described or specifiedin terms of their binding affinity to a P2Y10 polypeptide of the presentinvention. Representative binding affinities include those with adissociation constant or Kd less than 5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵M, 1×10⁻⁵M, 5×10⁻⁶M, 1×10⁻⁶M, 5×10⁻⁷M,1×10⁻⁷M, 5×10⁻⁸M, 1×10⁻⁸ M, 5×10⁻⁹M, 1×10⁻⁹ M, 5×10⁻¹⁰ M, 1×10⁻¹⁰ M,5×10¹¹ M, 1×10¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 1×10 ⁻¹³ M, 5×10⁻¹⁴M, 1×10⁻¹⁴ M, 5×10⁻¹⁵ M, or 1×10⁻¹⁵ M.

The present invention also provides antibodies that competitivelyinhibit binding of a ligand to an epitope of the present invention asdetermined by any method known in the art for determining competitivebinding, for example, the immunoassays described herein. Inrepresentative embodiments, the antibody competitively inhibits bindingto the epitope by at least 95%, at least 90%, at least 85%, at least80%, at least 75%, at least 70%, at least 60%, or at least 50%.

An antibody of the present invention includes derivatives that aremodified, i.e., by the covalent attachment of any type of molecule tothe antibody such that covalent attachment does not prevent the antibodyfrom generating an anti-idiotypic response. For example, but not by wayof limitation, an antibody derivative includes antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications can be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, a derivative can contain one or more non-classicalamino acids.

An antibody of the present invention can exert a stimulating or blockingeffect on an antigen. More specifically, by binding an antigen, anantibody of the present invention can block other molecules from bindingto or associating with the antigen. The blocking can be a stericconsequence of binding the antigen. The blocking effect can have theoverall effect of modulating one or more cellular processes bypreventing association of other molecules with the antigen or via thebinding event itself. Alternatively, the binding event can have astimulating effect on one or more cellular processes as a result of thebinding event itself or by making interactions between the antigen andanother molecule more feasible and/or more productive that is normallythe case.

III.A. Polyclonal Antibodies

The antibodies of the present invention can comprise polyclonalantibodies. Methods of preparing polyclonal antibodies are known to theskilled artisan (Harlow et al., Antibodies: A Laboratory Manual, 2^(nd)ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA(1988)). In one method, a preparation of a P2Y10 polypeptide is preparedand purified to render it substantially free of natural contaminants.Such a preparation is then introduced into an animal in order to producepolyclonal antisera of greater specific activity. For example, a P2Y10polypeptide of the present invention can be administered to various hostanimals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. The administration of a P2Y10 polypeptide of thepresent invention can entail one or more injections of an immunizingagent and, if desired, an adjuvant. Various adjuvants can be used toincrease the immunological response, depending on the host species, andinclude but are not limited to, Freund's (complete and incomplete),mineral gels such as aluminum hydroxide, surface active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are known to those or ordinary skill in the art.For the purposes of the present invention, “immunizing agent” is definedas a P2Y10 polypeptide of the present invention, including fragments,variants, and/or derivatives thereof, in addition to fusions withheterologous polypeptides and other forms of the polypeptides describedherein.

In some embodiments, the immunizing agent and/or adjuvant is injectedinto the subject (for example a mammal) by multiple subcutaneous orintraperitoneal injections, though they may also be administeredintramuscularly, and/or through IV injection. The immunizing agent caninclude polypeptides of the present invention or a fusion protein orvariants thereof. Depending on the nature of the polypeptides (i.e.,percent hydrophobicity, percent hydrophilicity, stability, net charge,isoelectric point etc.), it might be useful to conjugate the immunizingagent to a protein known to be immunogenic in the mammal beingimmunized. Such a conjugation can include either chemical conjugation byderivitizing active chemical functional groups to both the polypeptideof the present invention and the immunogenic protein such that acovalent bond is formed, or through fusion-protein based methodology, orother methods known to those of ordinary skill in the art. Examples ofsuch immunogenic proteins include, but are not limited to keyhole limpethemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybeantrypsin inhibitor. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas bacille Calmette-Guerin (BCG) and Corynebacterium parvum. Additionalexamples of adjuvants that can be employed include the MPL-TDM adjuvant(monophosphoryl lipid A, synthetic trehalose dicorynomycolate). Asuitable immunization protocol can be selected by one of ordinary skillin the art upon consideration of the present disclosure.

III.B. Monoclonal Antibodies

The antibodies of the present invention can comprise monoclonalantibodies. Monoclonal antibodies can be prepared using known hybridomamethods, as described herein and as is known to those of ordinary skillin the art (see, e.g., Köhler & Milstein, (1975) Nature 256:495; U.S.Pat. No. 4,376,110, Harlow et al., Antibodies: A Laboratory Manual,2^(nd) ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., USA (1988); Hammerling et al., Monoclonal Antibodies and T-CellHybridomas, Elsevier, New York, N.Y., USA, (1981) pp. 563-681; Köhler etal., (1976) Eur. J Immunol. 6:511; Köhler et al., (1976) Eur. J Immunol.6:292). Indeed hybridoma strain PTA-3975, which produces an antibody ofthe present invention, has been prepared and itself forms an aspect ofthe present invention.

Other examples of methods that can be employed for producing monoclonalantibodies include, but are not limited to, the human B-cell hybridomatechnique (Kosbor et al., (1983) Immunology Today 4:72; Cole et al.,(1983) Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridomatechnique (Cole et al. Monoclonal Antibodies And Cancer Therapy, Alan R.Liss, Inc., New York, N.Y., USA (1985) pp. 77-96). Such antibodies canbe of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and anysubclass thereof. A hybridoma producing a monoclonal antibody of thepresent invention can be cultivated in vitro or in vivo. Production ofhigh titers of monoclonal antibodies in vivo makes this a desirablemethod of production in some situations.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed below and elsewhere herein. In a non-limiting example, micecan be immunized with a P2Y10 polypeptide, or fragment thereof (e.g.,residues 171-191 of a P2Y10 polypeptide; SEQ ID NOs:3 and 4), of thepresent invention or a cell expressing such a polypeptide or fragment.Once an immune response is detected, e.g., antibodies specific for theantigen are detected in the mouse serum, the mouse spleen is harvestedand splenocytes isolated. The splenocytes are then fused by knowntechniques to any suitable myeloma cells, for example cells from a cellline available from the ATCC. Hybridomas are selected and cloned bylimited dilution. The hybridoma clones are then assayed by methods knownin the art for cells that secrete antibodies capable of binding apolypeptide of the present invention. Ascites fluid, which generallycontains high levels of antibodies, can be generated by immunizing micewith positive hybridoma clones.

Accordingly, in one aspect, the present invention provides a method ofgenerating a monoclonal antibody that specifically associates with aP2Y10 polypeptide, or a fragment thereof, as well as antibodies producedby the method, comprising: (i) culturing a hybridoma cell secreting anantibody of the present invention (e.g., an antibody that specificallyassociates with a P2Y10 polypeptide or a fragment thereof), optionallywherein the hybridoma is generated by fusing splenocytes isolated from amouse immunized with an antigen of the present invention (e.g. a P2Y10polypeptide or a peptide comprising residues 171-191 of P2Y10; SEQ IDNOS:3 and 4) with myeloma cells; and (ii) screening the hybridomasresulting from the fusion for hybridoma clones that secrete an antibodythat specifically associates with a P2Y10 polypeptide or a fragmentthereof.

A variety of methods exist in the art for the production of monoclonalantibodies and thus, the present invention is not limited solely totheir production in hydridomas. For example, monoclonal antibodies ofthe present invention can be made by employing recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. In this context, theterm “monoclonal antibody” refers to an antibody derived from a singleeukaryotic, phage, or prokaryotic clone. DNA encoding a monoclonalantibody of the present invention can be readily isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of murine antibodies, or such chains from human, humanized,or other sources). The hydridoma cells of the present invention (e.g.PTA-3975) can serve as a source of such DNA. Once isolated, the DNA canbe placed into expression vectors, which are then transformed into hostcells such as Simian COS cells, Chinese hamster ovary (CHO) cells, ormyeloma cells that do not otherwise produce immunoglobulin protein, toobtain the synthesis of monoclonal antibodies in the recombinant hostcells. The DNA also can be modified, for example, by substituting thecoding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences (U.S. Pat. No. 4,816,567;Morrison, (1985) Science 229:1202) or by covalently joining to theimmunoglobulin coding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptidecan be substituted for the constant domains of an antibody of thepresent invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the present invention to createa chimeric bivalent antibody.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of a hybridoma and recombinantly, asdescribed above, as well as phage display technologies, or a combinationthereof. For example, monoclonal antibodies can be produced usinghybridoma techniques, including those known in the art and taught, forexample, in Harlow et al. Antibodies: A Laboratory Manual, 2^(nd) ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA(1988); Hammerling et al., in: Monoclonal Antibodies and T-CellHybridomas, Elsevier, New York, N.Y., USA, (1981) pp. 563-681. The term“monoclonal antibody” as used herein is not limited to antibodiesproduced through hybridoma technology. Rather, the term “monoclonalantibody” broadly refers to an antibody that is derived from a singleclone, including any eukaryotic, prokaryotic, or phage clone, and notthe method by which it is produced.

Antibody fragments that recognize specific epitopes (e.g., residues171-191 of a P2Y10 polypeptide; SEQ ID NOS:3 and 4) can be generated byknown techniques. For example, Fab and F(ab')₂ fragments of the presentinvention can be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). F(ab′)₂ fragments contain avariable region, a light chain constant region and a CH1 domain of theheavy chain.

III.C. Human and Humanized Antibodies

Humanized antibodies form an aspect of the present invention (e.g., ahumanized anti-P2Y10 antibody). Humanized antibodies are antibodymolecules from non-human species that bind a desired antigen andcomprising one or more complementarity determining regions (CDRs) fromthe non-human species as well as a framework region from a humanimmunoglobulin molecule. Often, framework residues in the humanframework regions can be substituted with the corresponding residue froma CDR donor antibody to alter, and preferably improve, antigen binding.These framework substitutions can be identified by methods known in theart, e.g., by modeling of the interactions of the CDR and frameworkresidues to identify framework residues important for antigen bindingand sequence comparison to identify unusual framework residues atparticular positions (see, e.g., U.S. Pat. No. 5,585,089; Riechmann etal., (1988) Nature 332:323). Antibodies can be humanized using a varietyof techniques known in the art including, for example, CDR-grafting (EP239,400; PCT Publication WO 91/09967; U.S. Pat. Nos. 5,225,539;5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP519,596; Padlan, (1991) Molecular Immunology 28(4/5):489-498; Studnickaet al., (1994) Protein Engineering 7(6):805-814; Ropuska et al. (1994)Proc. Natl. Acad. Sci. U.S.A. 91:969-973), and chain shuffling (U.S.Pat. No. 5,565,332), each of which is incorporated herein by reference.

Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source that is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following known methods (Jones et al., (1986)Nature 321:522-525 (1986); Reichmann et al., (1988) Nature 332:323-327;Verhoeven et al., (1988) Science 239:1534-1536) by substituting rodentCDRs or CDR sequences for the corresponding sequences of a humanantibody. Accordingly, such “humanized” antibodies are chimericantibodies (U.S. Pat. No. 4,816,567), wherein substantially less than anintact human variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies aresometimes human antibodies in which some CDR residues and possible someFR residues are substituted from analogous sites in rodent antibodies.

In general, a humanized antibody comprises substantially all of at leastone, and typically two, variable domains, in which all or substantiallyall of the CDR regions correspond to those of a non-human immunoglobulinand all or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody can alsocomprise at least a portion of an immunoglobulin constant region (Fc),typically that of a human immunoglobulin (see Jones et al., (1986)Nature 321:522-525; Riechmann et al., (1988) Nature 332:323-329 andPresta, (1992) Curr. Opin. Struct. Biol. 2:593-596).

Completely human antibodies are particularly desirable for therapeutictreatment of human subjects. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedherein using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTPublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741. Other techniques are alsoavailable for the preparation of human monoclonal antibodies (Cole etal. Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, New York,N.Y., USA (1985); and Boemer et al., (1991) J. Immunol. 147(1):86-95).

Human antibodies can also be produced using transgenic mice that areincapable of expressing functional endogenous immunoglobulins, but canexpress human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes can be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion can be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes can be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring, which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the present invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonberg& Huszar, (1995) Int. Rev. Immunol. 13:65-93. See also PCT PublicationsWO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No.0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825;5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598,all of which are incorporated herein by reference. In addition,companies such as Abgenix, Inc. (Fremont, Calif., USA), Genpharm (SanJose, Calif., USA), and Medarex, Inc. (Princeton, N.J., USA) can beengaged to provide human antibodies directed against a selected antigenusing technology similar to that described herein.

Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and creation of an antibody repertoire.This approach is described, for example, in U.S. Pat. Nos. 5,545,807;5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,106, and in thefollowing scientific publications: Marks et al., (1992) Biotechnol.10:779-783; Lonberg et al., (1994) Nature 368:856-859; Fishwild et al.,(1996) Nature Biotechnol. 14:845-51; Neuberger, (1996) NatureBiotechnol. 14:826; Lonberg & Huszer, (1995) Intern. Rev. Immunol.13:65-93.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et a, (1988) Bio/technology12:899-903).

III.D. Monospecific/Monovalent Antibodies

The antibodies of the present invention can be monovalent antibodies.Methods for preparing monovalent antibodies are known in the art. Forexample, one method involves recombinant expression of immunoglobulinlight chain and modified heavy chain. The heavy chain is generallytruncated at a point in the Fc region so as to prevent heavy chaincrosslinking. Alternatively, the relevant cysteine residues aresubstituted with another amino acid residue or are deleted so as toprevent crosslinking.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart.

III.E. Bispecific/Bivalent Antibodies

The antibodies of the present invention can also be bispecificantibodies. Bispecific antibodies are monoclonal antibodies (e.g., humanor humanized monoclonal antibodies) that have binding specificities forat least two different antigens. In a bispecific antibody of the presentinvention, one of the binding specificities can be directed a P2Y10polypeptide of the present invention, the other can be for any otherantigen (e.g., a T lymphocyte-surface protein, receptor, receptorsubunit, tissue-specific antigen, virally derived protein, virallyencoded envelope protein, bacterially derived protein, interleukin,etc.).

Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities (Milstein& Cuello, (1983) Nature 305:537-539). Because of the random assortmentof immunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of ten different antibody molecules, ofwhich only one has the correct bispecific structure. The purification ofthe correct molecule can be accomplished by affinity chromatographysteps. Similar procedures are disclosed in PCT Publication WO 93/08829and in Traunecker et al., (1991) EMBO J 10:3655-3659.

Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion can be with an immunoglobulinheavy-chain constant domain, comprising at least part of the hinge, CH2,and CH3 regions. It is somteimes desirable to have the first heavy-chainconstant region (CH1) containing the site necessary for light-chainbinding present in at least one of the fusions. DNAs encoding theimmunoglobulin heavy-chain fusions and, if desired, the immunoglobulinlight chain, are inserted into separate expression vectors, and areco-transformed into a suitable host organism. For further details ongenerating bispecific antibodies see, e.g., Suresh et al., (1986) Meth.Enzymol. 121:210.

III.F. Anti-Idiotype Antibodies

Antibodies to a P2Y10 polypeptide, or fragment thereof (e.g., SEQ IDNOS:3 and 4), can, in turn, be utilized to generate anti-idiotypeantibodies that “mimic” a P2Y10 polypeptide or P2Y10 polypeptidefragment using techniques known to those skilled in the art (see, e.g.,Greenspan & Bona, (1989) FASEB J 7(5):437-444; and Nissinoff, (1991) JImmunol. 147(8):2429-2438). For example, antibodies that specificallyassociate with, and inhibit or facilitate, an association of a P2Y10polypeptide, or a fragment thereof, with a ligand, can be used togenerate anti-idiotypes that “mimic” a P2Y10 binding domain and, as aconsequence, bind to and inhibit or augment the biological effect of theligand binding event. Neutralizing anti-idiotypes or Fab fragments ofsuch anti-idiotypes can be used in therapeutic regimens to neutralize aP2Y10 polypeptide ligand. For example, such anti-idiotypic antibodiescan be used to bind a P2Y10 polypeptide, or fragment thereof, of thepresent invention and/or to bind its ligands, and thereby block thepolypeptide and/or ligand's biological activity.

Such anti-idiotypic antibodies capable of binding a P2Y10 polypeptidecan be produced in a two-step procedure. Such a method makes use of thefact that antibodies are themselves antigens, and therefore, it ispossible to obtain an antibody that binds to a second antibody. Inaccordance with this method, protein-specific antibodies (e.g.P2Y10-specific antibodies) can be used to immunize an animal, forexample a mouse. The splenocytes of such an animal are then used toproduce hybridoma cells, and the hybridoma cells are screened toidentify clones that produce an antibody whose ability to bind to theprotein-specific antibody can be blocked by the polypeptide itself. Suchantibodies comprise anti-idiotypic antibodies to the protein-specificantibody and can be used to immunize an animal to induce formation offurther protein-specific antibodies.

III.G. Heteroconjugate Antibodies

Heteroconjugate antibodies are also contemplated by the presentinvention. Heteroconjugate antibodies comprise two covalently joinedantibodies. Such antibodies have, for example, been proposed fortargeting immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for the treatment of HIV infection (PCT Publications WO91/00360; WO 92/20373; and EP03089). It is contemplated that theantibodies can be prepared in vitro using known methods in syntheticprotein chemistry, including those involving crosslinking agents. Forexample, immunotoxins may be constructed using a disulfide exchangereaction or by forming a thioester bond. Examples of suitable reagentsfor this purpose include iminothiolate and methyl-4-mercaptobutyrimidateand those disclosed, for example, in U.S. Pat. No. 4,676,980.

IV. Polynucleotides Encoding Antibodies

The present invention further encompasses polynucleotides comprising anucleotide sequence encoding an antibody of the present invention andfragments thereof. The present invention also encompassespolynucleotides that hybridize under stringent or lower stringencyhybridization conditions to polynucleotides that encode an antibody ofthe present invention, for example an antibody that specifically bindsto a P2Y10 polypeptide or an antibody that binds to a fragment of aP2Y10 polypeptide comprising the amino acid sequence of residues 171-191of a P2Y10 polypeptide (i.e., SEQ ID NOS:3 and 4).

The polynucleotides can be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known to those of ordinaryskill in the art. For example, if the nucleotide sequence encoding anantibody is known, a polynucleotide encoding the antibody can beassembled from chemically synthesized oligonucleotides (e.g., asdescribed in Kutmeier et al., (1994) BioTechniques 17:242), which,briefly, involves the synthesis of overlapping oligonucleotidescontaining portions of the sequence encoding the antibody, annealing andligating of those oligonucleotides, and then amplification of theligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody of the presentinvention can be generated from a nucleic acid derived from a suitablesource. If a clone containing a nucleic acid encoding a particularantibody is not available, but the sequence of the antibody molecule isknown, a nucleic acid encoding the immunoglobulin can be chemicallysynthesized or obtained from a suitable source (e.g., an antibody cDNAlibrary, or a cDNA library generated from a nucleic acid, for examplepoly A+ RNA, isolated from any tissue or cells expressing the antibody,such as hybridoma cells selected to express an antibody of the presentinvention) by PCR amplification using synthetic primers hybridizable tothe 3′ and 5′ ends of the sequence or by cloning using anoligonucleotide probe specific for the particular gene sequence toidentify, e.g., a cDNA clone from a cDNA library that encodes theantibody. Amplified nucleic acids generated by PCR can then be clonedinto replicable cloning vectors using any method known in the art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody canbe manipulated using methods known in the art for the manipulation ofnucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al. Molecular Cloning: A Laboratory Manual, (3^(rd) ed.)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA(2001), and Current Protocols in Molecular Biology, (Ausubel et al.,eds.), Greene Publishing Associates and Wiley-Interscience, New York,N.Y., USA (2002), and discussion presented herein below), to generateantibodies having a different amino acid sequence, for example to createamino acid substitutions, deletions, and/or insertions.

In a specific embodiment, an amino acid sequence of the heavy and/orlight chain variable domains can be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that areknown in the art, e.g., by comparison to known amino acid sequences ofother heavy and light chain variable regions to determine a region(s) ofsequence hypervariability. Using routine recombinant DNA techniques, oneor more of the CDRs can be inserted within framework regions, e.g., intohuman framework regions to humanize a non-human antibody, as describedherein. The framework regions can be naturally occurring or consensusframework regions, (e.g., human framework regions (see, e.g., Chothia eta, (1998) J. Mol. Biol. 278:457-479 for a listing of representativehuman framework regions). A polynucleotide generated by the combinationof the framework regions and CDRs can encode an antibody thatspecifically binds a P2Y10 polypeptide or fragment thereof. As discussedherein, one or more amino acid substitutions can be made within theframework regions, and the amino acid substitutions improve binding ofthe antibody to its antigen. Additionally, such methods can be used tomake amino acid substitutions or deletions of one or more variableregion cysteine residues participating in an intrachain disulfide bondto generate an antibody molecule lacking one or more intrachaindisulfide bonds. Other alterations to a polynucleotide are encompassedby the present invention and will be known to those of ordinary skill ofthe art upon consideration of the present disclosure.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., (1984) Proc. Natl. Acad. Sci. USA81:851-855; Neuberger et al. (1984) Nature 312:604-608; Takeda et al.,(1985) Nature 314:452-454) by splicing genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can begenerated and employed in the present invention. As described herein, achimeric antibody is a molecule in which different portions are derivedfrom different animal species, such as those having a variable regionderived from a murine mAb and a human immunoglobulin constant region,e.g., humanized antibodies.

Alternatively, techniques for the production of single chain antibodiesare known (see, e.g., U.S. Pat. No. 4,946,778; Bird, (1988) Science242:423-42; Huston et al., (1988) Proc. Natl. Acad. Sci. U.S.A.85:5879-5883; and Ward et al., (1989) Nature 334:544-54) and can beadapted to produce single chain antibodies. Single chain antibodies canbe formed by linking the heavy and light chain fragments of the Fvregion via an amino acid bridge, resulting in a single chainpolypeptide. Techniques for the assembly of functional Fv fragments inE. coli can also be used (Skerra et al., (1988) Science 242:1038-1041).

A clone encoding an antibody of the present invention can be obtainedaccording to the methods described herein and known to those of ordinaryskill in the art.

V. Methods of Producing Antibodies of the Present Invention

The antibodies of the present invention can be produced by any methodknown in the art for the synthesis of antibodies, in particular, bychemical synthesis or by recombinant expression techniques. Thefollowing is presented in addition to the discussion herein aboveregarding antibody production.

V.A. Recombinant

Recombinant expression of an anti-P2Y10 antibody of the presentinvention, or a fragment, derivative or analog thereof, (e.g., a heavyor light chain of an antibody of the present invention or a single chainanti-P2Y10 antibody of the present invention), requires the constructionof an expression vector comprising a polynucleotide that encodes such anantibody. Once a polynucleotide encoding an antibody molecule or a heavyor light chain of an antibody, or portion thereof (for examplecomprising a heavy or light chain variable domain) of the presentinvention has been obtained, a vector for the production of the antibodymolecule can be produced by recombinant DNA technology using techniquesknown in the art. Methods for preparing a protein by expressing apolynucleotide containing an antibody-encoding nucleotide sequence aredescribed herein.

Methods known to those of ordinary skill in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The presentinvention, thus, encompasses replicable vectors comprising a nucleotidesequence encoding an antibody molecule of the present invention, or aheavy or light chain thereof, or a heavy or light chain variable domain,operably linked to a promoter. Such vectors can include the nucleotidesequence encoding the constant region of an antibody molecule (see,e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S.Pat. No. 5,122,464), and a variable domain of the antibody can be clonedinto such a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the present invention. Thus, thepresent invention comprises host cells containing a polynucleotideencoding an antibody of the present invention, or a heavy or light chainthereof, or a single chain antibody of the present invention, operablylinked to a heterologous promoter. In representative embodiments for theexpression of double-chained antibodies, vectors encoding both the heavyand light chains may be co-expressed in the host cell for expression ofthe entire immunoglobulin molecule, as detailed herein.

A variety of host-expression vector systems can be employed to expressan antibody molecule of the present invention. Such host-expressionsystems represent vehicles by which a coding sequence of interest can beproduced and subsequently purified, but also represent cells that may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the present invention insitu. These include but are not limited to microorganisms such asbacteria (e.g., E. coli, B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining antibody coding sequences; yeast (e.g., Saccharomyces,Pichia) transformed with recombinant yeast expression vectors containingantibody coding sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, (CaMV); tobacco mosaic virus,(TMV)) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Under some conditions it might be desirable that bacterialcells such as Escherichia coli, or eukaryotic cells are used for theexpression of a recombinant antibody molecule, especially for theexpression of whole recombinant antibody molecule. For example,mammalian cells such as Chinese hamster ovary cells (CHO), inconjunction with a vector such as the major intermediate early genepromoter element from human cytomegalovirus is an effective expressionsystem for antibodies (Foecking et al., (1986) Gene 45:101; Cockett etal., (1990) Bio/Technology 8:2).

In bacterial systems, a number of expression vectors can beadvantageously employed, depending upon the use intended for theantibody molecule being expressed. For example, when a large quantity ofsuch a protein is to be produced, for example for the generation of apharmaceutical composition comprising an antibody molecule (such ananti-P2Y10 antibody, as described herein), vectors that direct theexpression of high levels of fusion protein products that are readilypurified can be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., (1983) EMBO J.2:1791), in which the antibody coding sequence can be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, (1985)Nucleic Acids Res. 13:3101-3109; Van Heeke & Schuster, (1989) J Biol.Chem. 24:5503-5509); and the like. pGEX vectors can also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orFactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) can be used as a vector to express foreign genes. The virusgrows in Spodoptera frugiperda cells. The antibody coding sequence maybe cloned individually into non-essential regions (for example thepolyhedrin gene) of the virus and placed under control of an AcNPVpromoter (for example the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems canbe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest can be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene can then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (see, e.g., Logan &Shenk, (1984) Proc. Natl Acad. Sci. U.S.A. 81:355-359). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., (1987)Method Enzymol. 153:51-544).

In addition, a host cell strain can be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products canbe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells that possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product can be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, W138, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines that stably express theantibody molecule can be engineered. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells can beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines that express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems can be used. For example, the herpessimplex virus thymidine kinase (Wigler et al., (1977) Cell 11:223),hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski,(1992) Proc. Natl. Acad. Sci. U.S.A. 48:202), and adeninephosphoribosyltransferase (Lowy et al., (1980) Cell 22:817) genes can beemployed in tk-, hgprt- or aprt- cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., (1980) Proc. Natl. Acad. Sci. U.S.A. 77:357; O'Hare et al.,(1981) Proc. Natl. Acad. Sci. U.S.A. 78:1527); gpt, which confersresistance to mycophenolic acid (Mulligan & Berg (1981) Proc. Natl. AcadSci. U.S.A. 78:2072); neo, which confers resistance to theaminoglycoside G-418 (Clinical Pharmacy 12:488-505; Wu & Wu, (1991)Biotherapy 3:87-95; Tolstoshev, (1993) Ann. Rev. Pharmacol. Toxicol.32:573-596; Mulligan, (1993) Science 260:926-932; and Morgan & Anderson,(1993) Ann. Rev. Biochem. 62:191-217; TIB TECH 11(5):155-215, May,1993); and hygro, which confers resistance to hygromycin (Santerre etal., (1984) Gene 30:147). Methods known in the art of recombinant DNAtechnology can be applied to select the desired recombinant clone, andsuch methods are described, for example, in Current Protocols inMolecular Biology, (Ausubel et al., eds.), Greene Publishing Associatesand Wiley-Interscience, New York (2002); Kriegler, Gene Transfer andExpression, A Laboratory Manual, Stockton Press, New York, N.Y., USA(1990); Current Protocols in Human Genetics, (Dracopoli et al., eds.),John Wiley & Sons, New York, N.Y., USA (1994), Chapters 12 and 13; andColberre-Garapin et al., (1981) J. Mol. Biol. 150:1.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington & Hentschel, in DNA Cloning,vol. 3, Academic Press, New York, N.Y., USA (1987)). When a marker inthe vector system expressing antibody is amplifiable, an increase in thelevel of inhibitor present in culture of host cell will increase thenumber of copies of the marker gene. Since the amplified region isassociated with the antibody gene, production of the antibody will alsoincrease (Crouse et al., (1983) Mol. Cell. Biol. 3:257).

The host cell can be co-transfected with two expression vectors of thepresent invention, the first vector encoding a heavy chain-derivedpolypeptide and the second vector encoding a light chain-derivedpolypeptide. The two vectors can contain identical selectable markersthat enable equal expression of heavy and light chain polypeptides.Alternatively, a single vector can be used which encodes, and is capableof expressing, both heavy and light chain polypeptides. In suchsituations, the light chain should be placed before the heavy chain toavoid an excess of toxic free heavy chain (Proudfoot, (1986) Nature322:52; Kohler, (1980) Proc. Natl. Acad. Sci. U.S.A. 77:2197). Thecoding sequences for the heavy and light chains can comprise cDNA orgenomic DNA.

Once an antibody molecule of the present invention has been produced byan animal, chemically synthesized, or recombinantly expressed, it can bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

V.B. Phage Display Methods

An anti-P2Y10 antibody of the present invention can also be generatedusing various phage display methods known in the art. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles that carry the polynucleotide sequences encoding them.In a particular embodiment, such phage particles can be utilized todisplay antigen binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make an antibody of the present invention include thosedisclosed in Brinkman et al., (1995) J Immunol. Methods 182:41-50; Ameset al., (1995) J Immunol. Methods 184:177-186; Kettleborough et al.,(1994) Eur. J. Immunol. 24:952-958; Persic et al., (1997) Gene 187 9-18;Burton et al., (1994) Adv. Immunology 57:191-280; PCT PublicationsPCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO93/11236; WO 95/15982; WO 95/20401; and in U.S. Pat. Nos. 5,698,426;5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047;5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and5,969,108; each of which is incorporated herein by reference in itsentirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described herein. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTPublication WO 92/22324; Mullinax et al., (1992) BioTechniques12(6):864-869; and Sawai et al., (1995) AJRI 34:26-34; and Better et al.(1988) Science 240:1041-1043. Examples of techniques that can be used toproduce single-chain Fv's and antibodies include those described in U.S.Pat. Nos 4,946,778 and 5,258,498; Huston et al., (1991) Method Enzymol.203:46-88; Shu et al., (1993) Proc. Natl. Acad. Sci. USA 90:7995-7999;and Skerra et a, (1988) Science 240:1038-1040.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it can be desirable to use chimeric, humanized,or human antibodies. A chimeric antibody is a molecule in whichdifferent portions of the antibody are derived from different animalspecies, such as antibodies having a variable region derived from amurine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art (see,e.g., Morrison, (1985) Science 229:1202; Oi et al. (1986) BioTechniques4:214; Gillies et al., (1989) J. Immunol. Methods 125:191-202; EP171496; EP 173494; PCT Publications WO 8601533; WO 8702671; Boulianne etal., (1984) Nature 312:643; Neuberger et al., (1985) Nature 314:268;U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397).

VI. Hybridomas of the Present Invention

A hybridoma can be employed in the production of a monoclonal antibodyof the present invention. Indeed, in one aspect of the present inventionhybridoma strain PTA-3975 was prepared as described in the Examples.Hybridoma strain PTA-3975 produces an anti-P2Y10 antibody of the presentinvention. Similar hybridomas can be prepared and used to produce anantibody of the present invention. Representative methods of preparing ahybridoma for the production of a monoclonal antibody of the presentinvention follow.

In one example of a hybridoma-based method of producing an antibody, amouse, a humanized mouse, a mouse with a human immune system, hamster,or other appropriate host animal, is typically immunized with animmunizing agent to elicit lymphocytes that produce or are capable ofproducing antibodies that will specifically bind to the immunizingagent. Alternatively, the lymphocytes may be immunized in vitro.

The immunizing agent typically, but not necessarily, includespolypeptides of the present invention or a fusion protein thereof. Inone example, the immunizing agent comprises a P2Y10polypeptide-expressing cell (or a cell expressing a fragment thereof,such as P2Y10 residues 171-191; SEQ ID NOS:3 and 4). Such cells can becultured in any suitable tissue culture medium; for example Earle'smodified Eagle's medium supplemented with 10% fetal bovine serum(inactivated at about 56° C.), and supplemented with about 10 g/l ofnonessential amino acids, about 1,000 U/ml of penicillin, and about 100μg/ml of streptomycin. Generally, either peripheral blood lymphocytes(PBLs) are used if cells of human origin are desired, or spleen cells orlymph node cells are used if non-human mammalian sources are desired.The lymphocytes are then fused with an immortalized cell line using asuitable fusing agent, such as polyethylene glycol, to form a hybridomacell (Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, New York, N.Y., USA (1986), pp. 59-103). Immortalized cell linesare often transformed mammalian cells, particularly myeloma cells ofrodent, bovine and human origin. Often, rat or mouse myeloma cell linesare employed. The hybridoma cells can be cultured in a suitable culturemedium that can optionally comprise one or more substances that inhibitthe growth or survival of the unfused, immortalized cells. For example,if the parental cells lack the enzyme hypoxanthine guaninephosphoribosyl transferase (HGPRT or HPRT), the culture medium for thehybridomas typically will include hypoxanthine, aminopterin, andthymidine (“HAT medium”), which substances prevent the growth ofHGPRT-deficient cells.

In some embodiments, preferred immortalized cell lines are those thatfuse efficiently, support stable high level expression of antibody bythe selected antibody-producing cells, and are sensitive to a mediumsuch as HAT medium. Other useful immortalized cell lines are murinemyeloma lines, which can be obtained, for instance, from the SalkInstitute Cell Distribution Center, San Diego, Calif., USA and theAmerican Type Culture Collection, Manassas, Va., USA. Yet other usefulimmortalized cell lines are the parent myeloma cell lines as provided bythe ATCC. As noted and implied throughout the specification, humanmyeloma and mouse-human heteromyeloma cell lines also have beendescribed for the production of human monoclonal antibodies (see, e.g.,Kozbor, (1984) J Immunol. 133:3001; Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, Marcel Dekker, Inc., New York,N.Y., USA (1987) pp. 51-63).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against aP2Y10 polypeptide or a fragment thereof (such as P2Y10 residues171-191). The binding specificity of monoclonal antibodies produced bythe hybridoma cells can be determined, for example byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbant assay (ELISA).Such techniques are known in the art. The binding affinity of themonoclonal antibody can, for example, be determined by a Scatchardanalysis (see Munson & Pollart, (1980) Anal. Biochem. 107:220.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods(see, e.g., Goding, Monoclonal Antibodies: Principles and Practice,Academic Press, New York, N.Y., USA (1986) and/or Wands et al., (1981)Gastroenterology 80:225-232). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640.Alternatively, the hybridoma cells may be grown in vivo as ascites in amammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-sepharose, hydroxyapatite chromatography, gel exclusionchromatography, gel electrophoresis, dialysis, or affinitychromatography, all of which techniques will be known to those ofordinary skill in the art.

VII. Representative Applications

The modulators of the present invention, particularly a modulatorcomprising an anti-P2Y10 antibody, can be used in a variety ofapplications. A representative, but non-limiting, list of applicationsfor modulators of the present invention includes (a) activating aresting T lymphocyte; (b) inducing surface expression of P2Y10 on aresting T lymphocyte; (c) stimulating P2Y10 mRNA expression in a restingT lymphocyte; and (d) inhibiting proliferation of an activated Tlymphocyte.modulation of T lymphocyte activity and modulating theexpression of P2Y 10 on the surface of a T lymphocyte. Otherapplications include the purification, detection and targeting of aP2Y10 polypeptide, including both in vitro and in vivo diagnostic andtherapeutic methods. Further, the antibody modulators of the presentinvention can be used in immunoassays for qualitatively andquantitatively measuring expression levels and amounts of P2Y10 in abiological sample, such a population of PBMC's (see, e.g., Harlow etal., Antibodies: A Laboratory Manual, 2^(nd) ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., USA (1988)).

As discussed in more detail below, a modulator of the present invention,e.g., an anti-P2Y10 antibody, can be used either alone or in combinationwith other compositions. An antibody of the present invention canfurther be recombinantly fused to a heterologous polypeptide at the N-or C-terminus or chemically conjugated (including covalently andnon-covalently conjugations) to a polypeptide or other composition. Forexample, an antibody of the present invention can be recombinantly fusedor conjugated to molecules useful as labels in detection assays andeffector molecules such as heterologous polypeptides, drugs,radionucleotides, or toxins (see, e.g., PCT Publications WO 92/08495; WO91/14438; WO 89/12624; U.S. Pat. No. 5,314,995; and EP 396,387).

Further discussion of some representative applications of antibodymodulators of the present invention follows. It is noted that althoughthe following discussion is primarily directed to antibody modulators,the discussion is applicable mutatis mutandis to any modulator. It isimplicit in the following discussion that the various methods andapplications can be carried out in vivo or in vitro.

VII.A. Diagnostic Assays

An antibody, or a fragment thereof, of the present invention can be usedin a diagnostic assay to detect the presence or absence of, or toquantify, an amount of a P2Y10 polypeptide or fragment thereof in asample. Such a diagnostic assay can comprise at least two steps. Thefirst step can comprise contacting a sample with an antibody of thepresent invention, wherein the sample is a tissue (e.g., human, animal,etc.), biological fluid (e.g., blood, urine, sputum, semen, amnioticfluid, saliva, etc.), biological extract (e.g., tissue or cellularhomogenate, etc.), a protein microchip (see, e.g., Arenkov et al.,(2000) Anal. Biochem. 278(2):123-131), or a chromatography column, etc.A second step can comprise quantifying an amount of antibody bound tothe substrate. Alternatively, the method can optionally involve a stepof attaching the antibody, for example covalently, electrostatically, orreversibly, to a solid support, and a second step of contacting thebound antibody with the sample, as defined above and elsewhere herein.

Antibodies can also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

VII.B. Protein Purification

An antibody, or fragments thereof, of the present invention can be fusedto marker sequences, such as a peptide to facilitate purification of aprotein, such as a P2Y10 polypeptide or a fragment thereof. In someembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., Chatsworth,Calif., USA), among others, many of which are commercially available. Asdescribed in Gentz et al., (1989) Proc. Natl. Acad. Sci. U.S.A.86:821-824, for instance, hexa-histidine provides for convenientpurification of the fusion protein. Other peptide tags useful forpurification include, but are not limited to, the “HA” tag, whichcorresponds to an epitope derived from the influenza hemagglutininprotein (Wilson et al. (1984) Cell 37:767) and the FLAG® tag (Sigma, St.Louis, Mo., USA).

VII.C. Pharmaceutical Compositions

An antibody of the present invention, with or without a therapeuticagent conjugated to it, administered alone or in combination with acytotoxic factor, a cytokine or other biologically active moiety,including a small molecule, can be used as a therapeutic.

An anti-P2Y10 antibody, or a fragment thereof, can be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g., analpha-emitter, such as, ²¹³Bi. The terms “cytotoxin” and “cytotoxicagent” include any agent that is detrimental to cells. Examples ofcytotoxins and cytotoxic agents include paclitaxol, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologues thereof. Representative therapeutic agents include, but arenot limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, melphalan,carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),and anti-mitotic agents (e.g., vincristine and vinblastine).

Techniques for conjugating such therapeutic moiety to antibodies areknown, (see, e.g., Arnon et al., in Monoclonal Antibodies And CancerTherapy, (Reisfeld et al., eds.), Alan R. Liss, Inc., New York, N.Y.,USA (1985) pp. 243-56; Hellstrom et al., in Controlled Drug Delivery,(2^(nd) ed.), (Robinson et al., eds.), Marcel Dekker, Inc., new York,N.Y., USA (1987) pp. 623-53; Thorpe, in Monoclonal Antibodies '84:Biological And Clinical Applications, (Pinchera et al., eds.), (1985)pp. 475-506; Monoclonal Antibodies For Cancer Detection And Therapy,(Baldwin et al., eds), Academic Press, New York, N.Y., USA (1985) pp.303-16, and Thorpe et al., (1982) Immunol. Rev. 62:119-58).

As well as the use of an antibody of the present invention as atherapeutic agent, the present invention also encompasses anti-P2Y10antibodies, and fragments thereof, conjugated to a diagnostic agent. Theantibodies can be used diagnostically, for example, to monitor thedevelopment or progression of a tumor as part of a clinical testingprocedure, for example to determine the efficacy of a given treatmentregimen. Detection can optionally be facilitated by coupling theantibody to a detectable substance. Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, radioactive materials,positron emitting metals using various positron emission tomographies,and nonradioactive paramagnetic metal ions. The detectable substance canbe coupled or conjugated either directly to the antibody (or fragmentthereof) or indirectly, through an intermediate (such as, for example, alinker known in the art) using techniques known in the art. See, forexample, U.S. Pat. No. 4,741,900, disclosing metal ions that can beconjugated to antibodies for use as diagnostics according to the presentinvention. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;examples of suitable prosthetic group complexes includestreptavidin/biotin and avidin/biotin; examples of suitable fluorescentmaterials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

The present invention also provides pharmaceutical compositions that donot comprise antibodies. Such compositions comprise a therapeuticallyeffective amount of a modulator of the present invention, and apharmaceutically acceptable carrier. In a specific embodiment, the term“pharmaceutically acceptable” means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water and water-based formulations are desirablecarriers when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents. Thesecompositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations andthe like. The composition can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oral formulationcan include standard carriers such as pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, sodium saccharine, cellulose,magnesium carbonate, etc. Examples of suitable pharmaceutical carriersare described in Remington's Pharmaceutical Sciences, (Gennaro, ed.)20th ed., Mack Publishing, Easton, Pa., USA (2000). Such compositionswill contain a therapeutically effective amount of the modulator, forexample in purified form, together with a suitable amount of carrier soas to provide the form for proper administration to the subject. Theformulation should suit the mode of administration.

In one embodiment, a composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, a composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where a composition is to be administered by infusion, itcan be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where a composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the present invention can be formulated as neutral orsalt forms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the present invention which will beeffective in the treatment, inhibition and prevention of a disease ordisorder associated with aberrant expression and/or activity of apolypeptide of the present invention can be determined by standardclinical techniques. In addition, in vitro assays may optionally beemployed to help identify optimal dosage ranges. The precise dose to beemployed in the formulation will also depend on the route ofadministration, and the seriousness of the disease or disorder, andshould be decided according to the judgment of the practitioner and eachsubject's circumstances. Effective doses can be extrapolated fromdose-response curves derived from in vitro or animal model test systems.

For antibodies, the dosage administered to a subject is typically 0.1mg/kg to 100 mg/kg of the subject's body weight. Preferably, the dosageadministered to a subject is between 0.1 mg/kg and 20 mg/kg of thesubject's body weight, more preferably 1 mg/kg to 10 mg/kg of thesubject's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of thepresent invention might be reduced by enhancing uptake and tissuepenetration (e.g., into the brain) of the antibodies by modificationssuch as, for example, lipidation.

A pharmaceutical composition can be administered in conjunction with apharmaceutically acceptable carrier, diluent, or excipient, to achieveany of the above-described therapeutic uses and effects. Suchpharmaceutical compositions can comprise agonists, antagonists,activators or inhibitors. The compositions can be administered alone, orin combination with at least one other agent or reagent, such as astabilizing compound, which may be administered in any sterile,biocompatible pharmaceutical carrier, including, but not limited to,saline, buffered saline, dextrose, and water. The compositions may beadministered to a patient alone, or in combination with other agents,drugs, hormones, or biological response modifiers.

The pharmaceutical compositions for use in the present invention can beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, vaginal, orrectal means.

In addition to the active ingredients, the pharmaceutical compositionscan contain pharmaceutically acceptable/physiologically suitablecarriers or excipients comprising auxiliaries which facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Further details on techniques for formulation andadministration are provided in Remington's Pharmaceutical Sciences,(Gennaro, ed.) 20th ed., Mack Publishing, Easton, Pa., USA (2000).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

In addition, pharmaceutical preparations for oral use can be obtained bythe combination of active compounds with a solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropyl-methylcellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth, andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or a physiologically acceptable saltthereof, such as sodium alginate.

Dragee cores may be used in conjunction with physiologically suitablecoatings, such as concentrated sugar solutions, which may also containgum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for product identification, or tocharacterize the quantity of active compound, i.e., dosage.

Pharmaceutical preparations, which can be used orally, further includepush-fit capsules made of gelatin, as well as soft, scaled capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with fillers or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances, which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. In addition,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyloleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents that increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants or permeation agents(enhancers) that are appropriate to the particular barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

A pharmaceutical composition may be provided as a salt and can be formedwith many acids, including but not limited to, hydrochloric, sulfuric,acetic, lactic, tartaric, malic, succinic, and the like. Salts tend tobe more soluble in aqueous solvents, or other protonic solvents, thanare the corresponding free base forms. In other cases, the preferredpreparation may be a lyophilized powder which may contain any or all ofthe following: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, ata pH range of 4.5 to 5.5, combined with a buffer prior to use. After thepharmaceutical compositions have been prepared, they can be placed in anappropriate container and labeled for treatment of an indicatedcondition. For administration of a modulator, such labeling can includeguidance on the amount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose or amount is well within the capability of thoseskilled in the art. For any compound, the therapeutically effective dosecan be estimated initially either in cell culture assays, for example,using neoplastic cells, or in animal models, usually mice, rabbits,dogs, or pigs. The animal model may also be used to determine theappropriate concentration range and route of administration. Suchinformation can then be used and extrapolated to determine useful dosesand routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient (e.g., a modulator of the present invention) thatameliorates, reduces, diminishes, or eliminates the symptoms orcondition. Therapeutic efficacy and toxicity can be determined bystandard pharmaceutical procedures in cell cultures or in experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio of toxic to therapeutic effects is the therapeutic index,which can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedata obtained from cell culture assays and animal studies are used indetermining a range of dosages for human use. A representative dosagecontained in a pharmaceutical composition is within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, the sensitivity of the patient, and the route ofadministration.

The practitioner, who will consider the factors related to an individualrequiring treatment, will determine the exact dosage. Dosage andadministration are adjusted to provide sufficient levels of the activecomponent, or to maintain the desired effect. Factors which may be takeninto account include the severity of the individual's disease state; thegeneral health of the patient; the age, weight, and gender of thepatient; diet; time and frequency of administration; drugcombination(s); reaction sensitivities; and tolerance/response totherapy. As a general guide, long-acting pharmaceutical compositions maybe administered every 3 to 4 days, every week, or once every two weeks,depending on half-life and clearance rate of the particular formulation.Variations in these dosage levels can be adjusted using standardempirical routines for optimization, as is well understood in the art.

As a guide, normal dosage amounts may vary from 0.1 to 100,000micrograms (μg), up to a total dose of about 1 gram (g), depending uponthe route of administration. Guidance as to particular dosages andmethods of delivery is provided in the literature and is generallyavailable to practitioners in the art. Those skilled in the art willemploy different formulations for nucleotides than for proteins or theirinhibitors or activators. Similarly, the delivery of polynucleotides orpolypeptides will be specific to particular cells, conditions,locations, and the like.

The present invention also provides a pharmaceutical pack or kitcomprising one or more containers filled with one or more of theingredients of the pharmaceutical compositions of the present invention.Optionally a notice can be associated with such container(s) in the formprescribed by a governmental agency regulating the manufacture, use orsale of pharmaceuticals or biological products, which notice reflectsapproval by the agency of manufacture, use or sale for humanadministration. Such a notice can also provide guidance on how to usethe pack or kit.

VII.D. P2Y10 Modulators

The present invention broadly encompasses modulators of P2Y10. Suchmodulators can act as agonists or antagonists. A P2Y10 modulator can beemployed in the various methods of the present invention. Suchmodulators can comprise for example, one, or a combination of, apolypeptide of variable length (including antibodies and fusionproteins) and a small molecule.

In one aspect of the present invention, a P2Y10 modulator thatassociates specifically with a P2Y10 polypeptide, e.g., an anti-P2Y10antibody, is described. Such a modulator has one or more propertiesselected from the group consisting of: (a) the ability to activate aresting T lymphocyte upon association with a P2Y10 polypeptide; (b) theability to cause expression of a P2Y10 polypeptide on the surface of aresting T lymphocyte upon association with a P2Y10 polypeptide; (c) theability to stimulate expression of mRNA encoding a P2Y10 polypeptide ina resting T lymphocyte upon association with a P2Y10 polypeptide; and(d) the ability to inhibit proliferation of an activated T lymphocyteupon association with a P2Y10 polypeptide.

A modulator can comprise any type of chemical entity, such as a proteinof any size, a small molecule or an antibody. An example of a modulatorthat is an antibody is presented herein, notably in the Examples. Justas there is no limitation on whether a modulator augments or inhibitsP2Y10 or P2Y10-mediated activity, there is no limitation on themechanism by which a modulator of P2Y10 acheives such an effect. Forexample, as further described herein, a modulator might block a ligand(e.g., an interleukin or other molecule secreted from, or present in, amixture of PBMC's) from associating with a P2Y10 polypeptide. In anothercase, a modulator might inhibit a P2Y10 polypeptide from associatingwith another polypeptide expressed on the surface of a T lymphocyte. Inyet another case, a modulator might facilitate the association of aP2Y10 polypeptide with another polypeptide expressed on the surface of aT lymphocyte.

A modulator of the present invention can comprise a fusion protein. Asone of ordinary skill in the art will appreciate, and as discussedherein, a modulator of the present invention can be fused to apolypeptide sequence. For example, a modulator of the present inventioncan be fused with the constant domain of immunoglobulins (IgA, IgE, IgG,IgM), or portions thereof (CH1, CH2, CH3, or any combination thereof andportions thereof) resulting in chimeric polypeptides. Such fusionproteins can facilitate purification and can increase half-life in vivo.This has been shown for chimeric proteins consisting of the first twodomains of the human CD4-polypeptide and various domains of the constantregions of the heavy or light chains of mammalian immunoglobulins (see,e.g., EP 394,827; Traunecker et al., (1988) Nature 331:84-86). Enhanceddelivery of an antigen across the epithelial barrier to the immunesystem has been demonstrated for antigens (e.g., insulin) conjugated toan FcRn binding partner such as IgG or Fc fragments (see, e.g., PCTPublication WO 96/22024 and PCT Publication WO 99/04813). IgG Fusionproteins that have a disulfide-linked dimeric structure due to the IgGportion disulfide bonds have also been found to be more efficient inbinding and neutralizing other molecules than monomeric polypeptides orfragments thereof alone (see, e.g., Fountoulakis et al., (1995) J.Biochem. 270:3958-3964). In one example, nucleic acids encoding amodulator can also be recombined with a gene of interest as an epitopetag (e.g., the hemagglutinin (HA) tag or FLAG® tag) to aid in detectionand purification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., (1991) Proc. Natl. Acad. Sci. USA 88:8972-8976). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix binding domain for the fusion. Extracts from cellsinfected with the recombinant vaccinia virus are loaded onto Ni²⁺nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Regardless of whether a given P2Y10 modulator is a small molecule,antibody or polypeptide, a modulator of the present invention can haveone or more properties selected from the group consisting of (a) theability to activate a resting T lymphocyte upon association with a P2Y10polypeptide; (b) the ability to induce expression of a P2Y10 polypeptideon the surface of a resting T lymphocyte upon association with a P2Y10polypeptide; (c) the ability to stimulate expression of mRNA encoding aP2Y10 polypeptide in a resting T lymphocyte upon association with aP2Y10 polypeptide; and (d) the ability to inhibit proliferation of anactivated T lymphocyte upon association with a P2Y10 polypeptide.

When a P2Y10 modulator is an antibody, the antibody can associatespecifically with P2Y10, for example residues 171-191 of a P2Y10polypeptide (SEQ ID NOs:3 and 4). Such an antibody can be produced by aknown or generated hybridoma strain such as PTA-3975, for example.

VII.D.1. Antibodies of the Present Invention as Modulators

An antibody of the present invention can bind an antigen comprising anantigenic epitope (e.g., a P2Y10 polypeptide), or a portion thereof(e.g., residues 171-191 of a P2Y10 polypeptide; SEQ ID NOS:3 and 4). AP2Y10 antibody of the present invention may act as an agonist or anantagonist (i.e., a modulator) of a P2Y10 polypeptide particularly, andmore generally as an agonist or antagonist of a biological activityinduced, mediated or inhibited by a P2Y10 polypeptide. The effect of theantibody, whether agonizing or antagonizing, can be a function of thestate (resting or activated) of a T lymphocyte on which the P2Y10 isexpressed or not expressed, as described herein.

Continuing, in one embodiment an antibody of the present invention canact as an agonist or an antagonist (i.e., a modulator) of an effectmediated by a P2Y10 polypeptide, such as T lymphocyte activation or theinhibition of the proliferation of T lymphocytes. For example, thepresent invention includes antibodies that may disrupt an interactionbetween a ligand or receptor and a P2Y10 polypeptide, either partiallyor fully. Such a disruption can be an agonizing or antagonizinginteraction and, in some embodiments, can depend on the state of a Tlymphocyte on the surface of which the P2Y10 polypeptide is expressed(e.g., activated or resting).

P2Y10 activation (e.g., signaling events initiated or perpetuated byP2Y10) can be determined by techniques described herein or otherwiseknown in the art. For example, P2Y10 activation can be determined bydetecting the phosphorylation state of P2Y10 or its substrate (e.g.,whether a given site, such as a tyrosine or serine/threonine residue isphosphorylated) by immunoprecipitation followed by western blotanalysis.

In another embodiment of the present invention, antibodies are providedthat may inhibit ligands binding to P2Y10 and thereby modulateP2Y10-mediated activity by at least 95%, at least 90%, at least 85%, atleast 80%, at least 75%, at least 70%, at least 60%, or at least 50% ofthe activity in absence of the antibody, thereby antagonizing oragonizing a biological activity.

When a P2Y10 modulator is an antibody, the antibody can specificallyassociate with a P2Y10 polypeptide, but need not associate with anyparticular portion of a P2Y10 polypeptide. In one example, an antibodycan associate with residues 171-191 of a P2Y10 polypeptide (SEQ ID NOS:3and 4).

An antibody modulator can be produced in any of a variety of ways, asdescribed herein. In one embodiment, an anti-P2Y10 antibody modulator isproduced by a hybridoma, such as hybridoma strain PTA-3975, which is arat hybridoma.

An antibody modulator of the present invention can have one or more ofthe following properties: (a) the ability to activate a resting Tlymphocyte; (b) the ability to induce surface expression of P2Y10 on aresting T lymphocyte; (c) the ability to stimulate P2Y10 mRNA expressionin a resting T lymphocyte; and (d) the ability to inhibit proliferationof an activated T lymphocyte. Each of these properties is discussed morefully herein below.

A modulator can have the ability to activate a resting T lymphocyte uponassociation with a P2Y10 polypeptide. T lymphocyte activation assays,such as those that measure T lymphocyte proliferation, are known in theart (see, e.g., Ledbetter et al., (1990) Blood 75:1531-39; Siefken etal., (1997) Cell. Immunol. 176:59-65). When a modulator is an anti-P2Y10antibody, association of an anti-P2Y10 antibody with a P2Y10 polypeptideis a natural result of exposing one to another, although such a bindingevent can be verified, if desired, using binding assays known in theart. The resting T lymphocyte can be isolated or can be a member of apopulation of PBMC's. Methods of preparing PBMC's from blood are known(e.g., by density gradient cetrifugation).

Another property a modulator can have is the ability to induceexpression of a P2Y10 polypeptide on the surface of a resting Tlymphocyte upon association with a P2Y10 polypeptide. Expression of aP2Y10 polypeptide on the surface of a T lymphocyte can be detected asdescribed herein, for example by FACS-based techniques. An assessment ofP2Y10 expression can be made, for example, by performing a FACS analysisof a resting T lymphocyte population to determine an amount of expressedP2Y10, followed by a FACS analysis of the population of T lymphocytesafter exposure to an anti-P2Y10 antibody.

Yet another property a modulator can have is the ability to stimulateexpression of mRNA encoding a P2Y10 polypeptide in a resting Tlymphocyte upon association with a P2Y10 polypeptide. Expression of mRNAencoding a P2Y10 polypeptide can be quantitated in any way, for example,by employing a binding assay or by employing a labeled anti-P2Y10antibody of the present invention.

A further property a modulator can have is the ability to inhibitproliferation of an activated T lymphocyte upon association with a P2Y10polypeptide. A T lymphocyte can be activated in any way, for example byexposing the T lymphocyte to phorbol ester, anti-CD3 antibodies,anti-CD28 antibodies or a combination of anti-CD3 and anti-CD28antibodies, as described herein. T lymphocyte proliferation can beconveniently measured by employing a tritium-labled thymidineincorporation assay, such as that described herein.

A modulator of the present invention can have one or a combination oftwo or more of the properties discussed above.

As noted herein, modulation can be performed in vivo or in vitro.Representative systems for both in vivo and in vitro modulationprotocols are provided herein.

VII.E. Modulation of a Biological Activity

A modulator of the present invention, such as an anti-P2Y10 antibody ofthe present invention, can be employed to modulate a biologicalactivity. The term “biological activity” includes any activity induced,mediated or inhibited by P2Y10 binding, for example, T lymphocyteactivation and/or proliferation, and upregulation and/or downregulationof an immune response. Modulation of a biological activity can beachieved by employing an anti-P2Y10 antibody alone, in combination withone or more additional therapeutics or drug moieties, or as a conjugatecomprising a therapeutic or drug moeity. A conjugate of the presentinvention can be used to modify a given biological response, and thetherapeutic agent or drug moiety is not to be construed as limited toclassical chemical therapeutic agents. For example, a drug moiety can bea protein or polypeptide possessing a desired biological activity. Suchproteins can include, for example, a toxin such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor, a-interferon, β-interferon, nerve growth factor,platelet derived growth factor, tissue plasminogen activator, anapoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (see, PCT PublicationWO 97/33899), AIM II (see, PCT Publication WO 97/34911), Fas Ligand(Takahashi et al., (1994) Int. Immunol. 6:1567-1574), VEGI (see, PCTPublication WO 99/23105), a thrombotic agent or an anti-angiogenicagent, e.g., angiostatin or endostatin; or, biological responsemodifiers such as, for example, lymphokines, interleukin-1 (IL-1),interleukin-2 (IL-2), interleukin-6 (IL-6), granulocyte macrophagecolony stimulating factor (GM-CSF), granulocyte colony stimulatingfactor (G-CSF), or other growth factors.

Modulation of a biological activity can encompass, for example, (a)activation of a resting T lymphocyte upon association with a P2Y10polypeptide; (b) inducing the expression of a P2Y 10 polypeptide on thesurface of a resting T lymphocyte upon association with a P2Y10polypeptide; (c) stimulating expression of mRNA encoding a P2Y10polypeptide in a resting T lymphocyte upon association with a P2Y10polypeptide; or (d) inhibiting proliferation of an activated Tlymphocyte upon association with a P2Y10 polypeptide.

Additionally, the modulation of a biological activity can be practicedin vivo or in vitro. When modulation is performed in vitro, themodulation can be performed as described in the Examples provided hereinbelow. When modulation is performed in vivo, the modulation can beperformed and detected in a subject, such as a mouse, rat or rabbit.

Examples of the modulation of some representative biological activitiesfollow.

VII.E.1. Method of Activating a Resting T Lymphocyte

In another aspect of the present invention, a modulator of the presentinvention can be employed to activate a resting T lymphocyte and can bepracticed in vitro or in vivo. As discussed herein, such a method may beemployed in the treatment of a condition such as cancer, a viralinfection, such as HIV, a bacterial infection, or any other condition inwhich it might be advantageous to augment an immune response.

In one embodiment of the method, a resting T lymphocyte is contactedwith a modulator of a P2Y10 polypeptide. A P2Y10 modulator can be amodulator as described herein, including, for example, an antibody. Asdemonstrated in the Examples and Tables, exposure of a resting Tlymphocyte to a modulator comprising an anti-P2Y10 antibody leads to Tcell proliferation. When a modulator is an antibody, it can be isolated,for example, from a hybridoma such as strain PTA-3975 and can associatewith a particular epitope on a P2Y10 polypeptide, such as residues171-191 (SEQ ID NOS:3 and 4). Alternatively, such an antibody can beisolated from a hybridoma prepared as described herein. Other modulatorscan be isolated from biological sources or prepared synthetically.

VII.E.2. Method of Inducing Expression of a P2Y10 Polypeptide on theSurface of a T Lymphocyte

In a further aspect of the present invention, a method of inducingexpression of a P2Y 10 polypeptide on the surface of a T lymphocyte isdisclosed. The method can be performed in vivo or in vitro. In oneembodiment of the method, a resting T lymphocyte is contacted with aP2Y10 modulator. The method may be useful, for example, in the treatmentof immune conditions in which P2Y10 expression is inhibited naturally orby a non-native inhibitor. The method may also be useful in thetreatment of disorders that are characterized by an unaceptably lowlevel of a T lymphocyte-dependent immune response. Such conditions caninclude cancer, viral infections such as HIV and bacterial infections,to name just a few.

Representative P2Y10 modulators are described herein and can be employedin the method. One example of a P2Y10 modulator is an anti-P2Y10antibody secreted by hybridoma strain PTW-3975. In this example, theantibody can bind specifically to residues 171-191 of P2Y10. Otherexamples of modulators include small molecules, proteins and nucleicacids.

The contacting can be carried out by any of a variety of approaches, thedetails of which can be dependent on the local environment of the Tlymphocyte. For example, T lymphocytes, which can be isolated or acomponent in a heterogeneous population of cells, can be washed with PBSand then incubated with a modulator under suitable conditions.

If and/or when it is desired to detect the expression of a P2Y10polypeptide on the surface of a T lymphocyte, a FACS-based approach canbe employed and can optionally form a component of the contacting. Byway of example, T lymphocytes can be washed with PBS containing FBS(FACS buffer) and then contacted with a modulator, such as an anti-P2Y10antibody of the present invention, under suitable conditions (e.g., 30minutes on ice). The cells can then be washed, labled with FITC or otheragent, contacted with anti-CD4 or anti-CD8 antibodies and analyzed byFACS.

VII.E.3. Method of Upregulating Transciption of P2Y10 mRNA in a TLymphocyte

In a related aspect, the present invention also encompasses a method ofupregulating transcription of P2Y10 mRNA in a T lymphocyte. The methodcomprises, in one embodiment, contacting a T lymphocyte with a modulatorof P2Y10. The method may be useful, for example, in the treatment ofimmune conditions in which P2Y10 expression is inhibited naturally or bya non-native inhibitor.

Representative P2Y10 modulators are described herein and can be employedin the method. One example of a P2Y10 modulator is an anti-P2Y10antibody secreted by hybridoma strain PTW-3975. In this example, theantibody can bind specifically to residues 171-191 of P2Y10. Otherexamples of modulators include small molecules, proteins and nucleicacids.

The contacting can be carried out by any of a variety of approaches, thedetails of which can be dependent on the local environment of the Tlymphocyte. For example, T lymphocytes, which can be isolated or acomponent in a heterogeneous population of cells, can be washed with PBSand then incubated with a modulator under suitable conditions.

When it is desired to detect a degree to which transcription of P2Y10mRNA is upregulated, any known method can be employed (see, e.g.,Sambrook et al. Molecular Cloning: A Laboratory Manual, (3^(rd) ed.)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA(2001), and Current Protocols in Molecular Biology, (Ausubel et al.,eds.), Greene Publishing Associates and Wiley-Interscience, New York,N.Y., USA (2002)), including various commercially available instrumentsand kits prepared for this purpose (e.g., TAQMAN, available from AppliedBiosystems, Foster City, USA).

VII.E.4. Method of Inhibiting Proliferation of an Activated T Lymphocyte

In still another aspect, the present invention comprises a method ofinhibiting the proliferation of an activated T lymphocyte. In oneembodiment the method comprises contacting an activated T lymphocytewith a modulator of a P2Y10 polypeptide. The method may also be usefulin the treatment of disorders that are characterized by an unaceptablyhigh level of a T lymphocyte-dependent immune response. Such conditionsinclude, for example, rheumatoid arthritis, psoriatic arthritis,multiple sclerosis, juvenile diabetes, asthma, inflammatory boweldisease (such as Crohn's disease and ulcerative colitus), pyodermagangrenum, lupus (systemic lupus erythematosis), myasthenia gravis,psoriasis, dermatitis, dermatomyositis; eczema, seborrhoea, pulmonaryinflammation, eye uveitis, hepatitis, Grave's disease, Hashimoto'sthyroiditis, autoimmune thyroiditis, Behcet's or Sjorgen's syndrome (dryeyes/mouth), pernicious or immunohaemolytic anaemia, atherosclerosis,Addison's disease (autoimmune disease of the adrenal glands), idiopathicadrenal insufficiency, autoimmune polyglandular disease (also known asautoimmune polyglandular syndrome), glomerulonephritis, scleroderma,morphea, lichen planus, viteligo (depigmentation of the skin), alopeciaareata, autoimmune alopecia, autoimmune hypopituatarism, Guillain-Barresyndrome, and alveolitis; T-cell mediated hypersensitivity diseases,including contact hypersensitivity, delayed-type hypersensitivity,contact dermatitis (including that due to poison ivy), uticaria, skinallergies, respiratory allergies (hayfever, allergic rhinitis) andgluten-sensitive enteropathy (Celiac disease); inflammatory diseasessuch as osteoarthritis, acute pancreatitis, chronic pancreatitis,asthma, acute respiratory distress syndrome, Sezary's syndrome andvascular diseases which have an inflammatory and or a proliferatorycomponent such as restenosis, stenosis and artherosclerosis.

Inflammatory or immune associated diseases or disorders also includes,but is not limited to: endocrine disorders, rheumatic disorders,collagen diseases, dermatologic disease, allergic disease, opthalmicdisease, respiratory disease, hematologic disease, gastrointestinaldisease, inflammatory disease, autoimmune disease, congenital adrenalhyperplasia, nonsuppurative thyroiditis, hypercalcemia associated withcancer, psoriatic arthritis, rheumatoid arthritis, including juvenilerheumatoid arthritis, ankylosing spondylitis, acute and subacutebursitis, acute nonspecific tenosynovitis, acute gouty arthritis,post-traumatic osteoarthritis, synovitis of osteoarthritis,epicondylitis, systemic lupus erythematosus, acute rheumatic carditis,pemphigus, bullous dermatitis herpetiformis, severe erythema multiforme,exfoliative dermatitis, psoriasis, seborrheic dermatitis, seasonal orperennial allergic rhinitis, serum sickness, bronchial asthma, contactdermatitis, atopic dermatitis, drug hypersensitivity reactions, allergicconjunctivitis, keratitis, herpes zoster ophthalmicus, iritis andiridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis,fulminating or disseminated pulmonary tuberculosis chemotherapy,idiopathic thrombocytopenic purpura in adults, secondarythrombocytopenia in adults, acquired (autoimmune) hemolytic anemia,leukemias and lymphomas in adults, acute leukemia of childhood,ulcerative colitis, regional enteritis, autoimmune vasculitis, multiplesclerosis, myasthenia gravis, anklyosing spondylitis, chronicobstructive pulmonary disease, solid organ transplant rejection, sepsis,and allergy.

A T lymphocyte can be activated in any of the range of activationmethods known to those of skill in the art and/or described herein. Forexample, a T lymphocyte can be activated by incubating T lymphocytes,which can be isolated or a component of heterogenous mixture such as apopulation of PBMC's, with, for example, phorbol myristate acetate (PMA)or anti-CD3 (Ledbetter et al., (1986) J Immunol. 136:3945-3952).

Representative P2Y10 modulators are described herein and can be employedin the method. One example of a P2Y10 modulator is an anti-P2Y10antibody secreted by hybridoma strain PTW-3975. In this example, theantibody can bind specifically to residues 171-191 of P2Y10. Otherexamples of modulators include small molecules, proteins and nucleicacids.

The contacting can be carried out by any of a variety of approaches, thedetails of which can be dependent on the local environment of the Tlymphocyte. For example, T lymphocytes, which can be isolated or acomponent in a heterogeneous population of cells, can be washed with PBSand then incubated with a modulator under suitable conditions.

A degree of T lymphocyte proliferation can be determined, for example,by monitoring uptake and/or incorporation of a detectable lable intolymphocytes undergoing clonal expansion. In one particular embodiment, adegree of proliferation can be identified by determing an amount of[³H]-thymidine incorporated into clonally expanding cells by liquidscintillation counting.

VII.F. Method of Modulating an Immune Response

The present invention also encompasses a method of modulating an immuneresponse. In one embodiment, the method comprises contacting a Tlymphocyte with a modulator of P2Y10. This method can be employed, forexample, to upregulate an immune response. Alternatively, a modulator ofP2Y10 can be contacted with an activated T lymphocyte in order toinhibit proliferation of T lymphocytes and thus downregulate an immuneresponse. The method can be carried out in vitro or in vivo. When themethod is performed in vivo, the modulator can be disposed in apharmaceutical composition as described herein and the contacting can beachieved by administering the pharmaceutical composition as describedherein.

As used herein, the term “immune response” encompasses any detectableresponse initiating with, or perpetuated by, a cell or molecule normallyhaving a physiological role in a mammalian immune system. Examples ofimmune response include T lmyphocyte activation, clonal expansion of Tlymphocytes, as well as secretion and/or binding of interleukins andother messengers.

Representative P2Y10 modulators are described herein and can be employedin the method. One example of a P2Y10 modulator is an anti-P2Y10antibody secreted by hybridoma strain PTW-3975. In this example, theantibody can bind specifically to residues 171-191 of P2Y10. Otherexamples of modulators include small molecules, proteins and nucleicacids.

The contacting can be carried out by any of a variety of approaches, thedetails of which can be dependent on the local environment of the Tlymphocyte. For example, T lymphocytes, which can be isolated or acomponent in a heterogeneous population of cells, can be washed with PBSand then incubated with a modulator under suitable conditions.

VII.G. Method of Treating an Immune Disease

The present invention can also be employed in the treatment of an immunedisease, as described herein above. For example, the present inventioncan be employed in situations in which a subject's immune system isdepressed and it is desired to enhance the activity and/or efficiency ofa subject's immune system. Such a situation might occur, for example, inthe case of a subject afflicted with a viral infection, such as HIV,cancer, a bacterial infection or other immune system-impairingcondition. Alternatively, the present invention can also be employed insitations in which a subject's immune system is upregulated and isoperating at a hypersensitive level. In such situations, it might bedesirable to downregulate the subject's immune response. Examples ofsuch conditions include, for example, rheumatoid arthritis, psoriaticarthritis, multiple sclerosis, juvenile diabetes, asthma, inflammatorybowel disease (such as Crohn's disease and ulcerative colitus), pyodermagangrenum, lupus (systemic lupus erythematosis), myasthenia gravis,psoriasis, dermatitis, dermatomyositis; eczema, seborrhoea, pulmonaryinflammation, eye uveitis, hepatitis, Grave's disease, Hashimoto'sthyroiditis, autoimmune thyroiditis, Behcet's or Sjorgen's syndrome (dryeyes/mouth), pernicious or immunohaemolytic anaemia, atherosclerosis,Addison's disease (autoimmune disease of the adrenal glands), idiopathicadrenal insufficiency, autoimmune polyglandular disease (also known asautoimmune polyglandular syndrome), glomerulonephritis, scleroderma,morphea, lichen planus, viteligo (depigmentation of the skin), alopeciaareata, autoimmune alopecia, autoimmune hypopituatarism, Guillain-Barresyndrome, and alveolitis; T-cell mediated hypersensitivity diseases,including contact hypersensitivity, delayed-type hypersensitivity,contact dermatitis (including that due to poison ivy), uticaria, skinallergies, respiratory allergies (hayfever, allergic rhinitis) andgluten-sensitive enteropathy (Celiac disease); inflammatory diseasessuch as osteoarthritis, acute pancreatitis, chronic pancreatitis,asthma, acute respiratory distress syndrome, Sezary's syndrome andvascular diseases which have an inflammatory and or a proliferatorycomponent such as restenosis, stenosis and artherosclerosis.

Inflammatory or immune associated diseases or disorders also includes,but is not limited to: endocrine disorders, rheumatic disorders,collagen diseases, dermatologic disease, allergic disease, opthalmicdisease, respiratory disease, hematologic disease, gastrointestinaldisease, inflammatory disease, autoimmune disease, congenital adrenalhyperplasia, nonsuppurative thyroiditis, hypercalcemia associated withcancer, psoriatic arthritis, rheumatoid arthritis, including juvenilerheumatoid arthritis, ankylosing spondylitis, acute and subacutebursitis, acute nonspecific tenosynovitis, acute gouty arthritis,post-traumatic osteoarthritis, synovitis of osteoarthritis,epicondylitis, systemic lupus erythematosus, acute rheumatic carditis,pemphigus, bullous dermatitis herpetiformis, severe erythema multiforme,exfoliative dermatitis, psoriasis, seborrheic dermatitis, seasonal orperennial allergic rhinitis, serum sickness, bronchial asthma, contactdermatitis, atopic dermatitis, drug hypersensitivity reactions, allergicconjunctivitis, keratitis, herpes zoster ophthalmicus, iritis andiridocyclitis, chorioretinitis, optic neuritis, symptomatic sarcoidosis,fulminating or disseminated pulmonary tuberculosis chemotherapy,idiopathic thrombocytopenic purpura in adults, secondarythrombocytopenia in adults, acquired (autoimmune) hemolytic anemia,leukemias and lymphomas in adults, acute leukemia of childhood,ulcerative colitis, regional enteritis, autoimmune vasculitis, multiplesclerosis, myasthenia gravis, anklyosing spondylitis, chronicobstructive pulmonary disease, solid organ transplant rejection, sepsis,and allergy.

Immune diseases associated with or secondary to other diseases can alsobe treated. Such diseases and conditions include recombinase activatinggene (RAG 1/2) deficiency, adenosine deaminase (ADA) deficiency,interleukin receptor chain (c) deficiency, Janus-associated kinase 3(JAK3) deficiency, reticular dysgenesis, DiGeorge syndrome, nudesyndrome, T cell receptor deficiency, MHC class II deficiency, TAP-2deficiency (MHC class I deficiency), ZAP70 tyrosine kinase deficiency,purine nucleotide phosphorylase (PNP) deficiency, X-linkedagammaglobulinemia (Bruton's tyrosine kinase deficiency), autosomalrecessive agammaglobulinemia:, Mu heavy chain deficiency, surrogatelight chain (5/14.1) deficiency), Hyper-IgM syndrome:, X-linked (CD40ligand deficiency), Ig heavy chain gene deletions, IgA deficiency,selective deficiency of IgG subclasses (with or without IgA deficiency),common variable immunodeficiency (CVID), antibody deficiency with normalimmunoglobulins, transient hypogammaglobulinemia of infancy, interferonreceptor (IFNGR1, IFNGR2) deficiency, Interleukin 12 and interleukin 12receptor deficiency, immunodeficiency with thymoma, Wiskott-Aldrichsyndrome (WAS protein deficiency), ataxia telangiectasia (ATMdeficiency), X-linked lymphoproliferative syndrome (SH2D 1A/SAPdeficiency), hyper IgE syndrome, Bloom syndrome, xeroderma pigmentosum,Fanconi anemia, ICF syndrome, Nijmegen breakage syndrome, Seckelsyndrome, Down syndrome (Trisomy 21), Turner syndrome, deletions orrings of chromosome 18 (18p- and 18q-), short-limbed skeletal dysplasia(short-limbed dwarfism), cartilage-hair hypoplasia (metaphysealchondroplasia), Schimke immuno-osseous dysplasia, Dubowitz syndrome,kyphomelic dysplasia with SCID, Mulibrey's nannism, growth retardation,facial anomalies and immunodeficiency, progeria (Hutchinson-Gilfordsyndrome), ectrodactyly-ectodermal dysplasia-clefting syndrome,immunodeficiency with absent thumbs, anosmia and ichthyosis, partialalbinism, dyskeratosis congenita, Netherton syndrome, anhidroticectodermal dysplasia, Papillon-Lefevre syndrome, congenital ichthyosis,acrodermatitis enteropathica, transcobalamin 2 deficiency, Type 1hereditary orotic aciduria, intractable diarrhea, abnormal facies,trichorrhexis and immunodeficiency, methylmalonic acidemia, biotindependent carboxylase deficiency, mannosidosis, glycogen storagedisease, type 1b, Chediak-Higashi syndrome, familial hypercatabolism,intestinal lymphangiectasia, chronic mucocutaneous candidiasis,hereditary or congenital hyposplenia or asplenia, Ivermark syndrome.

In one embodiment, the method comprises administering a modulator of aP2Y10 polypeptide to a subject, wherein the modulator has one or moreproperties selected from the group consisting of (a) the ability toactivate a resting T lymphocyte upon association with a P2Y10polypeptide; (b) the ability to induce expression of a P2Y10 polypeptideon the surface of a resting T lymphocyte upon association with a P2Y10polypeptide; (c) the ability to stimulate expression of mRNA encoding aP2Y10 polypeptide in a resting T lymphocyte upon association with aP2Y10 polypeptide; and (d) the ability to inhibit proliferation of anactivated T lymphocyte upon association with a P2Y10 polypeptide.

The recited properties (a)-(d) are described more fully herein above,which descriptions are applicable in toto to the context of the presentmethod.

Representative P2Y10 modulators are described herein and can be employedin the method. One example of a P2Y10 modulator is an anti-P2Y10antibody secreted by hybridoma strain PTW-3975. In this example, theantibody can bind specifically to residues 171-191 of P2Y10. Otherexamples of modulators include small molecules, proteins and nucleicacids. When a P2Y10 modulator is an antibody, the antibody can associatespecifically with residues 171-191 of a P2Y10 polypeptide (SEQ ID NOS:3and 4). Such an antibody can be produced by hybridoma strain PTA-3975,for example.

Various methods of administering a modulator to a subject are known andcan be employed in the present method. For example, a modulator can beadministered by injection and the injection can be made at any desirablesite, such as directly into the subject's circulatory system, ordirectly into an organ or other internal structure. Other methods ofadministration are described herein above.

VII.H. Method of Identifying a Modulator of a P2Y10 Polypeptide

Yet an additional aspect of the present invention relates to a method ofidentifying a modulator of a P2Y10 polypeptide. The method can beemployed, for example, in a screening program to identify test moleculesthat exhibit P2Y10 modulating activity and therefore may be suitable forfurther development or characterization. In one embodiment, the methodcomprises (a) measuring the ability of a test molecule to bind to a P2Y10 polypeptide; and (b) measuring one or more of: (i) the ability of thetest molecule to activate a resting T lymphocyte; (ii) the ability ofthe test molecule to induce surface expression of P2Y10 on a Tlymphocyte; (iii) the ability of the test molecule to stimulate P2Y10mRNA expression in a T lymphocyte; and (iv) the ability of the testmolecule to inhibit proliferation of an activated T lymphocyte, wherebya test molecule is identified as a modulator if the test molecule bindsto a P2Y10 polypeptide and exhibits one or more abilities selected fromthe group consisting of (i) to (iv).

The ability of a test molecule to bind to a P2Y10 polypeptide can bemeasured using known binding assays. For example, a test molecule can bedetectably labeled (e.g., radiolabled or fluorescently labeled) andcontacted with a P2Y10 polypeptide. Unbound test molecule can then beremoved. The ability of the test molecule to bind to a P2Y10 polypeptidecan be quantitatively assessed based on the amount of label associatedwith the P2Y10 polypeptide. When assessing binding of a test compound toa P2Y10 polypeptide, the binding can be assessed relative to a desireddegree. That is, binding can be defined by one or more quantitivemeasurements as described.

Assays for T lymphocyte activation, surface expression of P2Y10 on a Tlymphocyte, P2Y10 mRNA expression and T lymphocyte proliferation (andinhibition of proliferation) are known and some of which are describedherein. Similar to an assessment of the binding of a test molecule to aP2Y10 polypeptide, the abilities described in (i) to (iv) can bequantitatively assessed relative to a desired set of criteria. Forexample, a given test compound can be said to possess an ability if itproduces an effect recited in (i) to (iv) to a degree that is above orbelow a selected level.

After performing the above analysis, a test molecule identified asbinding a P2Y10 polypeptide to a desired degree (which can be a functionof, for example, percent label bound or based on binding constants) andexhibiting one or more properties selected from the group consisting of(i) to (iv) above is identified as a modulator. Modulators identified byemploying the method can be employed in the various methods of thepresent invention.

EXAMPLES

The following Examples have been included to illustrate representativemodes of the present invention. Certain aspects of the followingExamples are described in terms of techniques and procedures found orcontemplated by the present inventors to work well in the practice ofthe invention. These Examples are exemplified through the use ofstandard laboratory practices of the inventors. In light of the presentdisclosure and the general level of skill in the art, those of ordinaryskill in the art will appreciate that the following Examples areintended to be exemplary only and that numerous changes, modificationsand alterations can be employed without departing from the spirit andscope of the invention.

Example 1 Experimental Procedures for Example 1 Generation of Anti-P2Y10Monoclonal Antibody (mAb)

An antigenic peptide corresponding to amino acids 171-191 of P2Y10 (SEQID NO:3) was synthesized with a carboxy-terminal cysteine forconjugation to KLH (peptide sequence: RSTDLNNNKSCFADLGYKQMNC; SEQ IDNO:4). The KLH-conjugated peptide was used to generate rat anti-P2Y10monoclonal antibody by immunizing rats, selecting hybridomas asdetermined by binding to BSA-conjugated antigenic peptide, hybridomacloning, antibody purification and isotyping using the general procedurepublished by Starling et al. for the production of rat monoclonalantibodies (Starling et al., (1996) Eur. J Immunol. 26:738-746). Theanti-P2Y10 rat hybridoma was deposited with the ATCC Patent Depository(Accession No. PTA-3975, Jan. 11, 2001).

Activation of T Cells

Either anti-CD3 (G19-4, Genetic Systems Corporation, Seattle, Wash.,USA; see Ledbetter et al., (1986) J. Immunol 136:3945-3952), anti-P2Y10mAb, or IgG control at 1 μg/mL were immobilized on the surface of96-well flat-bottom plates (Nalge Nunc, Rochester, N.Y., USA, Cat#149026) at 4° C. overnight. After washing the plates three times withPBS, human peripheral blood T cells at 2×10⁵ cells/well were added. Somewells also received anti-CD28 (4 μg/mL, mAb9.3, Fred Hutchinson CancerResearch Center, Seattle, Wash., see Martin et al., (1986) J Immunol.136:3282-3287), and the cells were incubated at 37° C. for 4 days.Alternatively, phorbol ester (phorbol 12-myristate 13-acetate, Sigma,St. Louis, Mo., USA, Cat # P8139) at a concentration of 100 nM was addedto resting T-cells for 30 mins at 37° C.

T-Cell Proliferation Assay

Peripheral blood mononuclear cells containing T lymphocytes wereactivated as described above (some wells also contained anti-P2Y10 at 20μg/mL). After stimulation at 37° C. for 3 days, [³H]-thymidine (NEN) ata concentration of 5 μCi/mL was added. After an additional 12 hours at37° C., cells were harvested and the amount of tritium incorporationmeasured by liquid scintillation counting.

RT-PCR

Total RNA from T cells were isolated using TRIzol (Gibco BRL, Cat#15596), and RT-PCR performed using Gibco's two-step system(SUPERSCRIPTII RNase H reverse transcriptase, Gibco BRL, Cat # 18064,and eLONGase enzymes mix, Cat # 10480). To measure P2Y10 transcription,the following primers were used: CATCTGCTTCACTCCCTATCA (forward; SEQ IDNO:5) and CATTGATGAACCACTCTCCT (reverse; SEQ ID NO:6). The ampliconresulting from the RT-PCR using these primers corresponds to nucleotides759-1011 (253 bp) of P2Y10. For GAPDH, the following primers were used:TTAGCACCCCTGGCCAAGG (forward; SEQ ID NO:7) and CTTACTCCTTGGAGGCCATG(reverse; SEQ ID NO:8).

Detection of Surface Expression of P2Y10 on T Cells by FACS Analysis

Cells were washed with PBS containing FBS (FACS buffer) and then stainedby adding anti-P2Y10 mAb (1:100 dilution in FACS buffer) for 30 min onice. Cells were washed twice with cold FACS buffer followed by additionof mouse anti-rat IgG-FITC (Jackson ImmunoResearch, West Grove, Pa.,USA, Cat # 212-096-104) as secondary staining (1:200 dilution) andco-stained with anti-hCD4-PE (Becton Dickinson, 347327, 1:200 dilution),or anti-hCD8-PE (Becton Dickinson, Palo Alto, Calif., USA, Cat # 340046,1:200 dilution) for 15 mins. After washing twice with cold FACS buffer,cells were resuspended in 0.4 ml cold FACS buffer. FACS analysisperformed on a FACSort (Becton Dickinson, Palo Alto, Calif., USA).

Results and Discussion of Example 1

Using the peptide corresponding to amino acids 171-191 of P2Y10 asantigen, a rat anti-P2Y10 monoclonal antibody was identified andisolated. The anti-P2Y10 rat hybridoma has been deposited to the ATCCPatent Depository (No. PTA-3975, Jan. 11, 2001).

The anti-P2Y10 mAb is specific in that it only recognizes a single bandof 40 kD molecular weight on Western blots of B cell lysatescorresponding to the predicted MW for P2Y10 (see FIG. 1).

Using the anti-P2Y10 mAb, the effect of T cell activation on the surfaceexpression of P2Y10 was investigated by FACS analysis. As shown in Table1, both CD4+ and CD8+ T cells showed pronounced increases in P2Y10surface expression when stimulated with either anti-CD-3 alone oranti-CD-3/anti-CD-28 co-stimulation. Phorbol ester (PMA) stimulationalso induced P2Y10 surface expression, but to a smaller extent. Surfaceexpression of P2Y10 concomitant with stimulation of T cells suggests arole of P2Y10 in activated T cells. An additional interestingobservation was obtained in T cells incubated with the anti-P2Y10 mAb,with the antibody itself appearing to stimulate its own surfaceexpression.

Consistent with these findings, RT-PCR analysis of the amount of mRNAencoding P2Y10 that was induced in T cells stimulated by either phorbolester, anti-CD3, or anti-CD3/CD28 (see FIG. 2, GAPDH levels are shown asa control for the amount of mRNA). Little message was detected inunstimulated cells. Treatment of the cells with anti-P2Y10 mAb alsostimulated P2Y10 mRNA levels. These findings indicate that thestimulation of surface expression of P2Y10 results from stimulation ofmRNA encoding P2Y10.

Since P2Y10 expression is coincident with activation and stimulation ofT cells, the effect of anti-P2Y10 mAb on T cell proliferation wasinvestigated. As shown in Table 2, anti-CD3 and anti-CD3/CD28stimulation of T cells resulted in a robust proliferation of T cells asmeasured by [³H]-thymidine. Interestingly, the presence of theanti-P2Y10 mAb inhibited the proliferation induced by both anti-CD3 andanti-CD3/CD28 stimulation. Interestingly, the anti-P2Y10 mAb alonestimulated T cell proliferation. Consistent with these effects beingmediated by binding to P2Y10, a rat IgG isotype control showed littleinhibition of T cell proliferation as compared to the anti-P2Y10 mAb(Table 3). Moreover, anti-P2Y10 mAb which had been complexed with theantigenic peptide (amino acids 171-191 of P2Y10) greatly reduced theinhibition of T cell proliferation, thus demonstrating that the effectis P2Y10-specific.

These results are especially intriguing in that the modulation of P2Y 10by the anti-P2Y10 mAb has opposing effects depending on the state of theT cell. Indeed, resting T lymphocytes are stimulated to proliferate whentreated with the anti-P2Y10 mAb alone, and this stimulation regulatesits own expression. P2Y10 expression is enhanced upon stimulation of Tcells with anti-CD3 or anti-CD3/CD28, but the proliferation of cells isinhibited by the anti-P2Y10 mAb.

Although it is not the inventors' desire to be bound to any theory ofoperation, the following remarks are submitted. It is unclear howmodulation of P2Y10 function by the anti-P2Y10 mAb would regulate thesedisparate functions. Activation of T cells is a complex processinvolving a number of receptors including the T cell receptor (TCR),CD3, CD28, LFA-1, CD4, CD2 and CD45R. T cell recognition of an antigenicpeptide-MHC complex on an antigen-presenting cell results either inactivation and clonal expansion or in a state of nonresponsivenesscalled clonal anergy. Whether clonal expansion or clonal anergy ensuesis determined by the presence or absence of a co-stimulatory signal suchas that provided by the CD28/B7 interaction. If a resting T_(H) cellreceives the TCR-mediated signal in the absence of a suitableco-stimulatory signal, then the TH cell will become anergic. P2Y10 mayplay a role in the signaling through one or more of these co-stimulatoryreceptors and regulate the pathways toward expansion versus anergy.

It is also unclear whether the anti-P2Y10 mAb blocks the function ofP2Y10 or whether it activates P2Y10 (i.e., an agonistic response). Sincethe ligand for P2Y10 is unknown, it is difficult to determine the effectof the anti-mAb on the receptor. It may be possible to find a ligand bytransfecting cells with recombinant P2Y10 along with a promiscuous Gprotein such as G_(α15), then screen chemical libraries or tissuehomogenates to ascertain whether any P2Y10-dependent cell signaling canbe detected. Alternatively, the ability of chemical libraries or tissuehomogenates to induce binding of non-hydrolyzable GTP-[γ-S] cell lysatesfrom P2Y10-transfected cells may also be used to screen for ligands.Once an agonist for P2Y10 is identified, then the effect of theanti-P2Y10 mAb on the agonist-induced signaling could be measured todetermine whether the mAb blocks the function of the agonist. Evenwithout an agonistic ligand, it may be possible to test the effect ofthe anti-P2Y10 mAb itself in these systems to monitor for agonistic orantagonistic activity on baseline effects.

Even without knowing whether the anti-P2Y10 mAb antagonizes or agonizesthe function of P2Y 10, the present results indicate that a modulator ofP2Y 10 function may have uses in many human disorders. Indeed,modulation of T cell function would have a broad range of possibleindications. Disorders that could benefit from a P2Y10 modulator whichdampens T cell-dependent immune responses would include rheumatoidarthritis, psoriasis, inflammatory bowel disease, asthma, multiplesclerosis, chronic obstructive pulmonary disorder, systemic lupuserythematosus, and tissue/organ transplant rejection. Disorders thatcould benefit from a P2Y10 modulator that heightens T cell-dependentimmune responses would include cancer, viral infections such as HIV, andbacterial infections. TABLE 1 P2Y10 surface expression on stimulated Tcells as measured by FACS Treatment CD4+ cells CD8+ cells Unstimulated1.8%^(a) 1.1%^(b) Phorbol ester (PMA) 3.4% 1.4% anti-CD3 8.7% 4.9%anti-CD3/anti-CD28 6.9% 5.5% anti-P2Y10 9.8% 9.0%^(a)The percentage of CD4-positive cells that were alsoanti-P2Y10-positive.^(b)The percentage of CD4-positive cells that were alsoanti-P2Y10-positive.

TABLE 2 Effect of Anti-P2Y10 on T cell Proliferation Proliferation RatioStimulation (unstimulated = 1)^(a) anti-CD3 8.0 anti-CD3 + anti-P2Y102.6 anti-CD3/anti-CD28 5.7 anti-CD3/anti-CD28 + anti-P2Y10 2.3anti-P2Y10 6.4^(a)amount of [³H]-thymidine incorporation in stimulated cells dividedby the amount in unstimulated cells.

TABLE 3 Effect of Antigenic Peptide on Anti-P2Y10 Inhibition ofAnti-CD3-induced T cell Proliferation Treatment Inhibition^(a) Rat IgGcontrol  7% anti-P2Y10 65% Anti-P2Y10 + antigenic peptide 17%^(a)Percent inhibition of the amount of [³H]-thymidine incorporationinto anti-CD3-stimulated T cells.

REFERENCES

The references cited in the specification are incorporated herein byreference to the extent that they supplement, explain, provide abackground for or teach methodology, techniques and/or compositionsemployed herein. All cited patents, including patent applications, andpublications referred to in this application are herein expresslyincorporated by reference. Also expressly incorporated herein byreference are the contents of all citations of GenBank accessionnumbers, LocusID, and other computer database listings, as well as thecontents of the Sequence Listing associated herewith.

It will be understood that various details of the invention may bechanged without departing from the scope of the invention. Furthermore,the foregoing description is for the purpose of illustration only.

1.-5. (canceled)
 6. A method of identifying a modulator of a P2Y10polypeptide comprising (a) measuring the ability of a test molecule tobind to a P2Y10 polypeptide; and (b) measuring one or more of: (i) theability of said test molecule to activate a resting T lymphocyte; (ii)the ability of said test molecule to induce surface expression of P2Y10on a T lymphocyte; (iii) the ability of said test molecule to stimulateP2Y10 mRNA expression in a T lymphocyte; and (iv) the ability of saidtest molecule to inhibit proliferation of an activated T lymphocyte;whereby a test molecule is identified as a modulator if the testmolecule binds to a P2Y10 polypeptide and exhibits one or more abilitiesselected from the group consisting of (i) to (iv).
 7. The method ofclaim 6, wherein the modulator associates specifically with residues171-191 of a P2Y10 polypeptide (SEQ ID NO:3).
 8. The method of claim 6,wherein the modulator is an antibody.
 9. The method of claim 8, whereinthe antibody is produced by hybridoma strain PTA-3975.
 10. (canceled)